Methods for cell culturing

Abstract

The present invention relates to a novel composition for cultivating animal biomass in vitro. Corresponding methods are also provided herein. Further, this invention relates to a cellular composite for cultivating animal biomass in vitro, wherein the cellular composite comprises a cellular substrate and the composition of the invention. Corresponding methods for generating the cellular composite are also provided herein. Finally, a method for cultivating animal biomass in vitro in serum-free or reduced-serum conditions using the composition and cellular substrate of the invention are also provided.

Description

[0001] METHODS FOR CELL CULTURING

[0002] FIELD OF INVENTION

[0003] The present invention relates to a novel composition for cultivating animal biomass in vitro. Corresponding methods are also provided herein. Further, this invention relates to a cellular composite for cultivating animal biomass in vitro, wherein the cellular composite comprises a cellular substrate and the composition of the invention. Corresponding methods for generating the cellular composite are also provided herein. Finally, a method for cultivating animal biomass in vitro in serum-free or reduced-serum conditions using the composition and cellular substrate of the invention are also provided.

[0004] BACKGROUND

[0005] Due to environmental and ethical concerns that arise from the process of conventional animal production, there is a global pressure from consumers for the provision of alternatives to farmed animal products such as meat, skin and hide. A substitute to farmed animal products is cultivated biomass, which includes meat, skin and hide produced by culturing animal cells in vitro. In theory, cultivated biomass has the potential to eliminate many of the issues related to conventional animal production, however, it comes with technical challenges.

[0006] A major challenge for cultivated meat, skin and hide to be able to compete with farmed products is the cost to produce cultivated biomass. The growth medium has been estimated to contribute from 55 to 95 % of the overall cost of the cultivated process. Many researchers have attempted to optimise the growth medium, however, there exists a bottleneck as particular essential components, for example growth factors, remain expensive. Therefore, alternative optimisation strategies are required.

[0007] Typically, the growth medium utilises serum, such as fetal bovine serum (FBS) as it contains a variety of nutrients, growth factors, and proteins necessary for cell growth. However, the use of FBS is undesirable due to its cost, risks of pathogen contamination, source of contaminants, variability between batches, and ethics due to it being an animal-derived product. Although there have been some advancements with serum-free cell growth medium, it is typically not as effective as serum-based medium. Therefore, there remains a need to optimise cell growth in serum-free conditions using alternative techniques.

[0008] Additionally, cultivated biomass can be grown on scaffolds. However, there are numerous issues with scaffolds, such as the limitation of nutrient and oxygen diffusion in scaffolds of larger sizes, and the requirement for the scaffold to be biocompatible and / or edible. Therefore, it may be advantageous to carry out cultivation of animal biomass in a scaffold-free environment. The aim of the invention is to address some of the problems in the prior art, and provide new and improved methods, compositions, and cellular composites for use in cultivating animal biomass.

[0009] SUMMARY OF INVENTION

[0010] In one aspect, the invention provides a method for cultivating animal biomass in vitro, the method comprising:

[0011] (a) providing a composition comprising at least two cell populations, wherein the at least two cell populations are:

[0012] (a1) (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49:1 ; or

[0013] (a2) (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9:1 to about 99:1 ; and

[0014] (b) co-culturing the cells in the composition to produce a cultivated animal biomass.

[0015] Suitably, the method may be for cultivating meat in vitro, and the method may comprise:

[0016] (a) providing a composition, the composition comprising:

[0017] (i) myocytes and / or myocyte progenitor cells; and

[0018] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and

[0019] (b) co-culturing the cells in the composition to produce a cultivated meat product.

[0020] Suitably, the method may comprise:

[0021] (a) providing a composition, the composition comprising:

[0022] (i) fibroblasts and / or fibroblast progenitor cells; and

[0023] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9: 1 to about 99: 1 ; and

[0024] (b) co-culturing the cells in the composition to produce a cultivated animal biomass.

[0025] Suitably, the cultivated animal biomass may be cultivated meat, skin or hide.

[0026] Suitably, the co-culturing step may be performed in serum-free or reduced-serum conditions. Suitably, the co-culturing step may be performed for at least 24 hours.

[0027] Suitably, the cells of (i) and (ii) may be seeded on a substrate at a cell density of at least 10,000 cells / cm2, or at least 5 cells / mm3.

[0028] Suitably, the cultivated animal biomass may comprise a multicellular monolayer or a multicellular aggregate.

[0029] In another aspect, the invention provides a composition for cultivating animal biomass in vitro, wherein the composition comprises at least two cell populations, wherein the at least two cell populations are:

[0030] (a1) (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, wherein the ratio is from about 13:1 to about 49:1 ; or

[0031] (a2) (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9:1 to about 99:1.

[0032] Suitably, the composition may be for cultivating meat in vitro, wherein the composition may comprise:

[0033] (i) myocytes and / or myocyte progenitor cells; and

[0034] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, wherein the ratio is from about 13:1 to about 49:1.

[0035] Suitably, in any method or composition described herein, the ratio may be from about 19:1 to about 33:1.

[0036] Suitably, in any method or composition described herein, the ratio may be about 19:1.

[0037] Suitably, in any method or composition described herein, the myocytes and / or myocyte progenitor cells may be muscle satellite cells and / or myoblasts.

[0038] Suitably, the composition may comprise:

[0039] (i) fibroblasts and / or fibroblast progenitor cells; and

[0040] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9:1 to about 99:1.

[0041] Suitably, animal biomass may be cultivated meat, skin or hide.

[0042] Suitably, the ratio may be selected from the group consisting of:

[0043] (i) about 97:3 to about 93:7;

[0044] (ii) about 98:2 to about 91 :9;

[0045] (iii) about 95:5 to about 9: 1 ; and

[0046] (iv) about 99:1 to about 92:8.

[0047] Suitably, the ratio may be about 95:5.

[0048] Suitably, the composition may be a serum-free or reduced-serum composition.

[0049] Suitably, in any method or composition described herein, the cells of (ii) may be pre-adipocytes.

[0050] Suitably, in any method or composition described herein, the composition may comprise a cell culture medium.

[0051] Suitably, in any method or composition described herein, the composition may comprise serum.

[0052] Suitably, in any method or composition described herein, the composition may comprise a macromolecular crowding (MMC) agent.

[0053] Suitably, the MMC may be selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

[0054] Suitably, in any method or composition described herein, the MMC may be selected from: PEG8, PEG35, PVP40, PVP360 and / or carrageenan; or combinations thereof.

[0055] Suitably, in any method or composition described herein, the composition may comprise L- glutamine, L-alanyl-L-glutamine dipeptide, a natural and / or synthetic peptide growth factor, a neuregulin, a morphogenic protein, a vitamin, a carrier molecule, an amino alcohol, a trace metal and / or a lipid, or an analogue thereof, or activator or inhibitor of their molecular pathways. Suitably, in any method or composition described herein, the composition may be scaffold-free.

[0056] In another aspect, the invention provides a cultivated animal biomass produced by the method of the invention.

[0057] In another aspect, the invention provides a cultivated meat product produced by the method of the invention.

[0058] In another aspect, the invention provides a method for cultivating meat in vitro, the method comprising:

[0059] (a) providing a composition, the composition comprising:

[0060] (i) myocytes and / or myocyte progenitor cells; and

[0061] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ;

[0062] (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0063] (c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product.

[0064] Suitably, the cellular substrate may further comprise adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

[0065] Suitably, the co-culturing step may be performed in serum-free or reduced-serum conditions.

[0066] In another aspect, the invention provides a cellular composite comprising:

[0067] (a) a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0068] (b) a composition the composition comprising:

[0069] (i) myocytes and / or myocyte progenitor cells; and

[0070] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13: 1 to about 49: 1 ; wherein the cells of (i) and (ii) in the composition of (b) are located on and / or in the substrate of (a). Suitably, the cellular substrate of (a) may further comprise adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

[0071] In another aspect, the invention provides a method for generating a cellular composite that is suitable for use in cultivating meat in vitro, the method comprising:

[0072] (a) providing a composition, the composition comprising:

[0073] (i) myocytes and / or myocyte progenitor cells; and

[0074] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and

[0075] (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells to generate the cellular composite.

[0076] Suitably, the cellular substrate may further comprise adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

[0077] Suitably, in the composites or methods described herein, the cellular composite may be a serum- free or reduced-serum cellular composite.

[0078] Suitably, in the composites or methods described herein, the cellular substrate may comprise fibroblasts.

[0079] Suitably, in the composites or methods described herein, the cellular substrate may comprise a cell monolayer or a tissue sheet.

[0080] Suitably, in the composites or methods described herein, the cells of the composition may be located on the cellular substrate.

[0081] Suitably, in the composites or methods described herein, the composite may be scaffold-free.

[0082] In another aspect, the invention provides a cellular composite produced by the method of the invention. In another aspect, the invention provides a method for cultivating meat in vitro in or reduced- serum conditions, the method comprising:

[0083] (a) providing a serum free serum-free or reduced-serum composition, the composition comprising:

[0084] (i) myocytes and / or myocyte progenitor cells; and

[0085] (ii) adipocytes and / or adipose progenitor cells;

[0086] (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0087] (c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product.

[0088] Suitably, the cellular substrate may further comprise adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

[0089] Suitably, in the compositions or methods described herein, the myocytes and / or myocyte progenitor cells may be muscle satellite cells and / or myoblasts.

[0090] Suitably, in the compositions or methods described herein, the adipocytes and / or adipose progenitor cells may be pre-adipocytes.

[0091] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0092] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0093] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect or example of the disclosure are to be understood to be applicable to any other aspect, or example described herein unless incompatible therewith.

[0094] Various aspects of the disclosure are described in further detail below. Brief description of the Figures

[0095] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0096] Figure 1 : Murine muscle:fat cell co-cultures have higher proliferation capacity. A) C2C12 murine myoblasts co-cultured with 3T3-F442A murine pre-adipocytes at different ratios (99:1 to 90:10; grey bars) up to 5 days in SFM showed different proliferation rates compared with control myoblast mono-cultures (white bars). B) The increased proliferation of C2C12 and 3T3-F442A cells at a 95:5 ratio was further improved when co-cultures were incubated with serum-free medium containing macromolecular crowding agents. Cell number, expressed as % of seeded cells, corresponded to average ± S.D. of three independent experiments (n =3; ** and ***, p = 0.01 and 0.001 , respectively).

[0097] Figure 2: Murine muscle:fat cell co-cultures enhance tissue biomass production. A) The proliferation of C2C12 murine myoblasts cultured alone for 14 days (white bars) in serum-free medium containing 20 g / L of polyvinylpyrrolidone 360,000 (+PVP) was significantly (4-fold) higher than SFM control (SFM), but lower than with serum-containing media (FBS). Proliferation was further improved in co-cultures of C2C12 and 3T3-F442A cells at a 95:5 ratio (grey bars) in all conditions tested, albeit not significantly. B) The amount of muscle tissue produced from C2C12 cells alone (white bars) in FBS and +PVP conditions was significantly higher than that in SFM (by 2.7- and 4.3-fold, respectively). Moreover, C2C12 cells co-cultured with 3T3-F442A cells at a 95:5 ratio (grey bars) showed to produce more biomass in all conditions tested, with SFM, FBS, and +PVP showing a 1.6-, 1.5-, and 1.1-fold increase over their corresponding mono-culture controls, respectively). Cell number and biomass production were normalised against values from monocultures in SFM and corresponded to average ± S.D. of three independent experiments (n =3; *, ** and ***, p = 0.05, 0.01 and 0.001 , respectively).

[0098] Figure 3: Porcine muscle:fat cell co-cultures enhance tissue biomass production, structural integrity, and recovery. Micrographs from tissues produced from porcine muscle cell mono-cultures (top row), fat cell mono-cultures (centre), or muscle:fat cell co-cultures (95:5 ratio) (bottom row) after 2 weeks growth in high serum, low serum, or serum-free media. Tissues produced in different conditions were subsequently recovered and imaged by photography with background elimination (insets) Scale bars: 200 pm (insets: 2 mm).

[0099] Figure 4: Schematic of the different methodologies to seed muscle and fat cells in coculture. Muscle and fat cells are seeded together and evenly distributed (homogeneous co- seeding), seeded at the same time but spatially separated (heterogeneous co-seeding), or seeded together but at different time points (sequential seedings). All muscle:fat co-cultures are then grown with the same culture media up to 21 days to allow for cells to proliferate and produce tissues.

[0100] Figure 5: Impact of different muscle and fat cell seeding methodologies on cell numbers during proliferation and tissue formation processes. The number of cells from co-cultures established using homogeneous co-seeding, heterogeneous co-seeding, and sequential seeding methods was analysed at day 2, 7, and 21 of culture, and compared with that from muscle monocultures. Cell number, expressed as % of seeded cells, corresponded to average ± S.D. of three independent experiments (n =3; ** and ***, p = 0.01 and 0.001 , respectively).

[0101] Figure 6: Porcine skin:fat cell co-cultures have higher proliferation capacity. Primary dermal fibroblasts isolated from porcine skin and primary pre-adipocytes isolated from porcine fat cocultured at different ratios (99:1 to 80:20; grey bars) up to 5 days in SFM showed different proliferation rates compared with control fibroblast mono-cultures (100:0 ratio; white bars) or control preadipocyte mono-cultures (0:100 ratio; black bar). The increased proliferation of fibroblasts and pre-adipocytes at a 97:3, 96:4, 95:5, 94:6, and 93:7 ratio was demonstrated by the significantly reduced cell doubling time in these co-cultures compared with fibroblast monocultures. Cultures with ratios of 90:10 to 0:100 showed significantly higher cell doubling time compared with fibroblast mono-cultures. Doubling time, expressed as % of 100:0 control, corresponded to average ± S.D. of seven independent experiments (n = 7; * and ***, p < 0.05 and 0.001 , respectively).

[0102] Figure 7: Porcine skin:fat cell co-cultures enhance tissue biomass production. The biomass of tissues produced from primary skin fibroblasts and primary pre-adipocytes co-cultured at different ratios (99:1 to 80:20) was compared with that from tissues produced from control fibroblast mono-cultures (100:0 ratio) or control preadipocyte mono-cultures (0:100 ratio) after 7 and 14 days (white and grey bars, respectively). A) As a general trend, total collagen content was higher in co-cultures compared with fibroblast or pre-adipocytes mono-cultures, with significant increases shown for 98:2- to 91 :9-ratio co-cultures at day 7, and for 95:5-ratio co-culture at day 14. B) Similarly, total protein content was higher in co-cultures compared with fibroblast or pre- adipocytes mono-cultures, with significant increases shown for 95:5- to 90:10-ratio co-cultures at day 7, and for 99:1- to 92:8-ratio co-cultures at day 14. Total collagen and protein production were normalised against values from fibroblast mono-cultures in SFM on day 7 or 14 and corresponded to average ± S.D. of four independent experiments (n = 4; * and #, p < 0.05 on day 7 and 14, respectively).

[0103] Figure 8: Schematic of the different methodologies to seed and co-culture muscle or muscle and fat cells on skin tissue substrates. Porcine muscle cells were seeded alone or in combination with fat cells (95:5 ratio) on skin tissue substrates, and co-cultured for two weeks prior to whole-tissue recovery and analysis.

[0104] Figure 9: Impact of skin tissue substrates on the production of tissues from muscle or muscle and fat cell co-cultures. Top row: Tissues produced from porcine muscle cells grown for 2 weeks alone and with trace-amounts of serum (left) or with serum-free media (SFM) (centre), or in combination with porcine fat cells (95:5 ratio) in SFM conditions. Bottom row: Skin tissues grown alone (left) or used as substrates for the co-culture of porcine muscle cells alone (centre) or in combination with porcine fat cells (95:5 ratio) in SFM. Scale bars: 100 pm.

[0105] DETAILED DESCRIPTION

[0106] The present invention is based on the inventors’ surprising finding that co-culturing of muscle cells or skin cells with fat cells results in an increased overall number of cells in the culture. Moreover, the present inventors have identified that this beneficial effect is observed when cells are cocultured after being seeded at a specific ratio. This specific ratio is from about 13:1 to about 49:1 for muscle:fat co-cultures, and from about 9:1 to about 99:1 for skin:fat co-cultures. As seen for example in Figure 1A and Figure 6, an increase in cell number is especially apparent when cells are co-cultured at a seeding ratio of about 19:1 (i.e. about 95:5).

[0107] The inventors have also shown that co-culturing of muscle cells with fat cells in serum-free media results in a decrease in cell number and biomass as compared to co-culturing of these cells in media comprising serum (for example fetal bovine serum (FBS)). However, unexpectedly the need for serum can be obviated by co-culturing of muscle cells with fat cells in serum-free media comprising a macromolecular crowding agent, such as PVP, as shown in Figure 2.

[0108] Additionally, the inventors have surprisingly shown that seeding muscle and fat cells at a ratio of from about 13:1 to about 49:1 onto a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells, and subsequent co-culture results in a substantial increase in overall biomass of obtained cellular composite in comparison to seeding muscle and fat cells onto a non-biological substrates, as demonstrated in Figure 9. The overall biomass is increased even when taking into account the additional fibroblasts and / or fibroblast progenitor cells of the cellular composite. As explained in the Examples section, the increased biomass is solely due to the cellular composites’ contribution as dry weight of muscle (M), fat (F) and fibroblasts and / or fibroblast progenitor cells (S) i.e. M+F+S is still higher (2.1 mg / cm2) than that of M+F cells and separate S cells combined (1.5-1.7 mg / cm2). A benefit of this is that the nutrient-dry and nutrient-wet weight conversion is higher, which means less feed is required for product cultivation, rendering the cultivation more cost and / or time effective. The inventors believe that the benefit of a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells is at least in part due to the presence of a fibroblast derived ECM in such a cellular substrate.

[0109] Moreover, the inventors have surprisingly shown that co-culturing of fibroblasts and / or fibroblast progenitor cells with fat cells at a ratio of from about 9:1 to about 99:1 results in substantially higher overall biomass in comparison with co-cultures made from fibroblasts and / or fibroblast progenitor cells alone, as demonstrated in Figure 7.

[0110] Furthermore, the inventors have surprisingly shown that when muscle and fat cells are seeded at a ratio from about 13:1 to about 49:1 onto a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells and co-cultured in serum-free media, the final product has a comparable biomass to when muscle and fat cells are seeded onto the same composite and co-cultured in media containing serum. Therefore, the presence of a skin substrate eliminates the requirement for serum during co-culture. This is highly advantageous as use of media containing serum, such as fetal bovine serum (FBS), in methods of cultivating meat is undesirable due to its cost, scalability, sustainability, risks of pathogen contamination, source of contaminants, variability between batches, and ethics, etc.

[0111] Method for cultivating animal biomass

[0112] In one aspect, the present invention provides a method for cultivating animal biomass. The aim of this method is to produce a cultivated animal biomass product.

[0113] In one example, the method may be for cultivating meat. The aim of this method is to produce a cultivated meat product.

[0114] In one example, the method may be for cultivating meat, skin or hide. The aim of this method is to produce a cultivated meat or leather product.

[0115] Suitably, the method may be an in vitro method.

[0116] The term “cultivating” as used herein refers to producing an animal biomass product (e.g. a meat product, a skin product, a hide product, or a leather product) in an artificial environment. The product is obtained by co-culturing cells. In one example, the co-culture is of (i) myocytes and / or myocyte progenitor cells, with (ii) adipocytes and / or adipose progenitor cells. Therefore, the animal biomass product (e.g. meat product) may comprise or consist of (i) myocytes and / or myocyte progenitor cells, and (ii) adipocytes and / or adipose progenitor cells.

[0117] In the present disclosure, where the context allows, myocytes and / or myocyte progenitor cells may be sometimes referred to as “cells of (i)”. Furthermore, in the present disclosure, myocytes and / or myocyte progenitor cells, and / or the cells they can differentiate into (for example myocytes and / or myocyte progenitor cells can differentiate to myocytes) may be referred to herein as “muscle cells”.

[0118] By the same token, in the present disclosure, where the context allows, adipocytes and / or adipose progenitor cells may be sometimes referred to as “cells of (ii)”. Furthermore, in the present disclosure adipocytes and / or adipose progenitor cells, and / or the cells they can differentiate into (for example adipose progenitor cells can differentiate to adipocytes) may be referred to herein as “fat cells”.

[0119] Other examples of fat and muscle cells are provided herein below. Accordingly, it can be said that the animal biomass product comprises or consists of muscle cells and fat cells.

[0120] In another example, the co-culture is of (i) fibroblasts and / or fibroblast progenitor cells, with (ii) adipocytes and / or adipose progenitor cells. Therefore, the animal biomass product (e.g. meat product, skin product, hide product, or leather product) may comprise or consist of (i) fibroblasts and / or fibroblast progenitor cells, and (ii) adipocytes and / or adipose progenitor cells.

[0121] In the present disclosure, where the context allows, fibroblasts and / or fibroblast progenitor cells may sometimes be referred to as “cells of (i)”. Furthermore, in the present disclosure, fibroblasts and / or fibroblast progenitor cells, and / or the cells they can differentiate into (for example fibroblasts and / or fibroblast progenitor cells can differentiate to fibroblasts) may be referred to herein as “skin cells”.

[0122] Adipocytes and / or adipose progenitor cells are described above. Other examples of fat and skin cells are provided herein below. Accordingly, it can be said that the animal biomass product comprises or consists of muscle cells and skin cells.

[0123] The term “animal biomass” refers to a composition comprising edible cells and / or tissues of animal origin. The edible cells may also be referred to as “animal-like cells”. The term “animal origin” refers to cells originally derived from but not directly obtained from an animal. A cell that is originally derived from but not directly obtained from an animal is, for example, a cell that has arisen as a result of a cell directly obtained from an animal undergoing cell division in cell culture. In another example, a cell that is originally derived from but not directly obtained from an animal is a cell that has been directly obtained from an animal and has undergone differentiation in cell culture. Accordingly, in the context of the present disclosure, animal-like cells are cells that have been produced in vitro by culturing a cell that itself was indirectly obtained from an animal, or a cell that was directly obtained from an animal. A cell that has been directly obtained from an animal may be physically removed from an animal (for example by a biopsy) or from animal tissues (for example from an animal carcass). Cells that are indirectly obtained from an animal may be from an established cell line or cell strain. Examples of animal biomass are meat, skin and hide.

[0124] Suitably, the animal biomass (e.g. meat) may have structural, compositional, or organoleptic properties (colour, taste and / or aroma) at least similar to meat obtained from an animal.

[0125] In the art, cultivated animal biomass includes cultivated meat, cultivated skin, cultivated hide, or cultivated leather.

[0126] Cultivated meat may also be referred to as cultured meat, healthy meat, slaughter-free meat, in vitro meat, lab-grown meat, cell-based meat, cellular meat, clean meat, artificial meat, and / or synthetic meat.

[0127] Cultivated skin may also be referred to as cultured skin, healthy skin, slaughter-free skin, in vitro skin, lab-grown skin, cell-based skin, cellular skin, clean skin, artificial skin, and / or synthetic skin.

[0128] Cultivated hide may also be referred to as cultured hide, healthy hide, slaughter-free hide, in vitro hide, lab-grown hide, cell-based hide, cellular hide, clean hide, artificial hide, and / or synthetic hide.

[0129] Cultivated leather may also be referred to as cultured leather, alternative leather, slaughter-free leather, in vitro leather, lab-grown leather, cell-based leather, cellular leather, clean leather, artificial leather, sustainable leather, and / or synthetic leather.

[0130] The terms “hide” and “leather” may be used interchangeably herein.

[0131] In one embodiment of the present invention, the cultivated animal biomass product may be a multicellular monolayer animal biomass product. In another embodiment of the present invention, the cultivated animal biomass product may be a multicellular aggregate.

[0132] The term “multicellular monolayer” as used herein describes a layer of cells which is one cell in thickness. A multicellular monolayer of the present invention may comprise two or more cell types. A multicellular monolayer of the present invention may be irregular in shape, but still only one cell thick. In some embodiment, the monolayer may be continuous or discontinuous. By continuous it is meant that the monolayer does not comprise or substantially does not comprise any holes. By discontinuous it is meant that the monolayer comprises areas of where there are no cells (i.e. holes).

[0133] The term “multicellular aggregate” as used herein, describes a cluster of cells which is more than one cell in thickness. Suitably, the multicellular aggregate may comprise two, three, four, five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, a hundred, or more layers of cells in thickness (i.e. it may be a multilayer of cells). The multicellular aggregate may be irregular in structure or a globule. Alternatively, the multicellular aggregate may be a multilayer cellular sheet. The multicellular aggregate may be the shape of for example a steak, a burger patty, a sausage, a cold meat cut, a portion of crackling, or a piece of skin.

[0134] In the context of the present disclosure, where reference is made to a “layer” of cells (for example myocytes and / or myocyte progenitor cells, adipocytes and / or adipose progenitor cells, fibroblasts, and / or fibroblast progenitor cells), the layer may be a monolayer, or multilayer.

[0135] The cultivated animal biomass may be heterogeneous in structure. The heterogeneity of the structure may be obtained from the two or more types of cells used in the method of cultivating animal biomass in vitro. For example, the cultivated animal biomass may have the appearance of fat marbled meat.

[0136] The method of cultivating animal biomass in vitro, comprises the step of:

[0137] (a) providing a composition comprising at least two cell populations, wherein the at least two cell populations are:

[0138] (a1) (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; or

[0139] (a2) (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells.

[0140] In one example, the method is a method for cultivating meat in vitro, wherein the composition comprises:

[0141] (i) myocytes and / or myocyte progenitor cells; and

[0142] (ii) adipocytes and / or adipose progenitor cells.

[0143] In another example, the method is a method of cultivating animal biomass wherein the composition comprises:

[0144] (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells.

[0145] In this example, the cultivated animal biomass may be cultivated meat, skin or hide.

[0146] The term “providing” as used herein means placing (for example by pipetting) the composition or cells of the composition into a cell culture vessel. It will be appreciated that the cells of (i) and (ii) may be provided simultaneously, for example as a mixture. Alternatively, the cells of (i) and (ii) may be provided simultaneously but not as a mixture, i.e. the cells of (i) and (ii) are provided from separate sources (for example pipettes), comprising the cells of (i) and (ii) separately. In such an embodiment, upon placement of the cells of (i) and (ii) the cells may get mixed or may remain separated for a period of time (for example several hours or days). Alternatively, the cells of (i) and (ii) may be provided sequentially. In such an embodiment, upon placement of the cells of (i) and (ii) the cells may get mixed or may remain separated for a period of time (for example several hours or days).

[0147] It will be appreciated that the cells of (i) and (ii) may form a composition once provided to the cell culture vessel.

[0148] The composition may comprise, consist of, or substantially consist of (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are cells of (i) and (ii).

[0149] In a suitable embodiment, the method comprises providing a composition comprising myocytes and adipocytes.

[0150] In a suitable embodiment, the method comprises providing a composition comprising myocyte progenitor cells and adipocytes.

[0151] In a suitable embodiment, the method comprises providing a composition comprising myocytes and adipose progenitor cells.

[0152] In a suitable embodiment, the method comprises providing a composition comprising myocyte progenitor cells and adipose progenitor cells.

[0153] In a suitable embodiment, the method comprises providing a composition comprising myocytes, adipocytes and adipose progenitor cells.

[0154] In a suitable embodiment, the method comprises providing a composition comprising myocytes, myocyte progenitor cells and adipocytes. In a suitable embodiment, the method comprises providing a composition comprising myocytes, myocyte progenitor cells, and adipose progenitor cells.

[0155] In a suitable embodiment, the method comprises providing a composition comprising myocyte progenitor cells, adipocytes, and adipose progenitor cells.

[0156] In a suitable embodiment, the method comprises providing a composition comprising myocytes, myocyte progenitor cells, adipocytes, and adipose progenitor cells.

[0157] In another example, the composition may comprise, consist of, or substantially consist of (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are cells of (i) and (ii).

[0158] In a suitable embodiment, the method comprises providing a composition comprising fibroblasts and adipocytes.

[0159] In a suitable embodiment, the method comprises providing a composition comprising fibroblast progenitor cells and adipocytes.

[0160] In a suitable embodiment, the method comprises providing a composition comprising fibroblast and adipose progenitor cells.

[0161] In a suitable embodiment, the method comprises providing a composition comprising fibroblast progenitor cells and adipose progenitor cells.

[0162] In a suitable embodiment, the method comprises providing a composition comprising fibroblasts, adipocytes and adipose progenitor cells.

[0163] In a suitable embodiment, the method comprises providing a composition comprising fibroblasts, fibroblast progenitor cells and adipocytes.

[0164] In a suitable embodiment, the method comprises providing a composition comprising fibroblasts, fibroblast progenitor cells, and adipose progenitor cells.

[0165] In a suitable embodiment, the method comprises providing a composition comprising fibroblast progenitor cells, adipocytes, and adipose progenitor cells.

[0166] In a suitable embodiment, the method comprises providing a composition comprising fibroblasts, fibroblast progenitor cells, adipocytes, and adipose progenitor cells.

[0167] The term “myocyte” as used herein refers to cells that have the ability to contract. There are at least three types of myocyte cells known, which are skeletal muscle myocyte, smooth muscle myocyte, and cardiac muscle myocyte. Myocytes may form myotubes and / or myofibrils. Accordingly, in the context of the present disclosure the term myocyte may also include myotubes and / or myofibrils. Suitably, the myocyte may be identified by expression of myoblast determination protein 1 (MYOD1), myogenin (MyoG), and lack of expression of paired box protein 7 (Pax7). Suitably, the myocytes in the form of myotubes and / or myofibrils may be identified by expression of) myogenin (MyoG) and myogenic factor 4 (MRF4).

[0168] The term “myocyte progenitor cell” refers to any cell that has the potential to differentiate into a myocyte. Suitably, the myocyte progenitor cell may be a stem cell, for example an induced pluripotent stem cell. The myocyte progenitor cell may have the ability to differentiate into a myocyte, multinucleated myotube and / or myotubular tissue without passing through some or any of the phases of the canonical pathway (a stem cel l / satel lite cell phase, and / or a myoblast phase) of muscle cell differentiation e.g., via a ‘synthetic’ pathway. For example, an induced pluripotent stem cell may differentiate into a myocyte, multinucleated myotubes, and / or myotubular tissue without passing through a satellite cell, and / or myoblast phase, or only passing through one of these phases.

[0169] Alternatively, the myocyte progenitor cell may have the potential to follow the canonical pathway of muscle cell differentiation i.e., stem cel l / satel lite cell phase, then a myoblast phase, then a myocyte phase.

[0170] Suitably, the myocyte progenitor cell may be a myoblast. The term “myoblast” as used herein refers to a cell that is committed to differentiating into a myocyte. As it will be appreciated by the person skilled in the art, myocytes can fuse to form multinucleated myotubes, and optionally further form muscle fibres and / or myotubular tissue. Myoblast cells can be identified by expression of the myoblast marker paired box protein 3 (Pax3), paired box protein 7 (Pax7), myogenic factor 5 (MYF5), and / or myoblast determination protein 1 (MYOD1). Suitably, the myoblast may be a porcine cell. Suitably, the myoblast may be a mouse cell (such as C2C12).

[0171] Suitably, the myocyte progenitor cell may be a muscle satellite cell (also referred to herein as a “satellite cell”). In vivo, a satellite cell is a quiescent muscle stem cell. Suitably, the satellite cell is positive for Pax7, Pax3, and / or MYF5 markers. Suitably, the satellite cell may be a porcine satellite cell.

[0172] The term “adipocyte” as used herein refers to a cell that has differentiated and become specialized in the synthesis and / or storage of fat. In vivo, adipocytes are derived from mesenchymal stem cells which give rise to adipocytes through adipogenesis. Suitably, the adipocyte may be selected from the group consisting of a white adipocyte, beige adipocyte, and brown adipocyte. An adipocyte marker includes adiponectin (ADIPOQ), peroxisome proliferator-activated receptor y (PPARy), fatty acid-binding protein 4 (FABP4), cytoplasmic glycerol-3-phosphate dehydrogenase (GPDH), Uncoupling Protein 1 (UCP1), and / or leptin (LEP), among others, and depending on adipocyte type.

[0173] The term “adipose progenitor cell” refers to a cell having the ability to differentiate into a mature adipocyte. Suitably the adipose progenitor cell may be selected from the group consisting of a pre-adipocyte cell, an adipose stem cell and a mesenchymal stem cell. Suitably, the adipose progenitor cell (such as a pre-adipocyte cell) may be positive for CD34, CD90 and CD271 and negative for CD31 , CD45, CD104b, CD105, and CD146. Suitably, the pre-adipocyte cell is a porcine pre-adipocyte cell. Suitably the pre-adipocyte cell is a mouse pre-adipocyte cell (such as with 3T3-F442A cell).

[0174] The terms “fibroblast” and “fibroblast progenitor cell” are defined elsewhere herein and apply equally to all aspects of the invention.

[0175] In one example, the cultivated animal biomass (e.g. cultivated meat) may comprise animal cells or tissues made from cells selected from the group consisting of myocyte progenitor cells, myocytes (for example in form of myotubes, myofibrils and / or muscle fibres), adipocytes (for example brown, beige, and / or white adipocytes) and adipose progenitor cells. Suitably, it can be said the cultivated animal biomass (e.g. cultivated meat) comprises muscle and fat. As it will be appreciated by the person skilled in the art, the cells selected from the group consisting of myocyte progenitor cells, myocytes, and muscle fibres give rise to the muscle component (i.e. muscle cells) of the cultivated animal biomass (e.g. cultivated meat), and the adipocytes and / or adipose progenitor cells give rise to the fat component (i.e. fat cells) of the cultivated animal biomass product (e.g. cultivated meat).

[0176] In another example, the cultivated animal biomass (e.g. cultivated meat, skin or hide) may comprise animal cells or tissues made from cells selected from the group consisting of fibroblast progenitor cells, fibroblasts, adipocytes (for example brown, beige, and / or white adipocytes) and adipose progenitor cells. Suitably, it can be said the cultivated animal biomass (e.g. cultivated meat, skin or hide) comprises skin and fat. As it will be appreciated by the person skilled in the art, the cells selected from the group consisting of fibroblast progenitor cells and fibroblasts give rise to the skin component (i.e. skin cells) of the cultivated animal biomass (e.g. cultivated meat, skin or hide), and the adipocytes and / or adipose progenitor cells give rise to the fat component (i.e. fat cells) of the cultivated animal biomass product (e.g. cultivated meat, skin or hide).

[0177] In another example, the cultivated animal biomass (e.g. cultivated meat, skin or hide) may comprise animal cells or tissues made from cells selected from the group consisting of myocyte progenitor cells, myocytes (for example in form of myotubes, myofibrils and / or muscle fibres), fibroblast progenitor cells, fibroblasts, adipocytes (for example brown, beige, and / or white adipocytes) and adipose progenitor cells. Suitably, it can be said the cultivated animal biomass (e.g. cultivated meat, skin or hide) comprises muscle, skin and fat. As it will be appreciated by the person skilled in the art, the cells selected from the group consisting of myocyte progenitor cells, myocytes, and muscle fibres give rise to the muscle component (i.e. muscle cells) of the cultivated animal biomass, the cells selected from the group consisting of fibroblast progenitor cells and fibroblasts give rise to the skin component (i.e. skin cells) of the cultivated animal biomass, and the adipocytes and / or adipose progenitor cells give rise to the fat component (i.e. fat cells) of the cultivated animal biomass product (e.g. cultivated meat, skin or hide).

[0178] In examples where the composition comprises:

[0179] (i) myocytes and / or myocyte progenitor cells; and

[0180] (ii) adipocytes and / or adipose progenitor cells, the cells in the composition may be provided at a ratio from about 13:1 to about 49:1 of myocytes and / or myocyte progenitor cells to adipocytes and / or adipose progenitor cells (i.e. cells of (i) to cells of (ii)). The term “ratio” refers to the number of myocytes and / or myocyte progenitor cells to adipocytes and / or adipose progenitor cells. For example, a ratio of 19:1 means that for every 19 myocytes and / or myocyte progenitor cells that are present in the composition, there is 1 adipocyte or adipose progenitor cell.

[0181] As it will be clear to a person of skill in the art, a ratio of 19:1 is equivalent to a ratio of 38:2, 57:3, 76:4, 95:5, and so on. Therefore, when myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells, are at a ratio of about 19:1 , about 38 myocytes and / or myocyte progenitor cells will be present and about 2 adipocytes and / or adipose progenitor cells will be present, and so on. Likewise, if about 12x106myocytes and / or myocyte progenitor cells are present, about 6x105adipocytes and / or adipose progenitor cells will be present in a ratio of 19:1.

[0182] A ratio can be presented in the form of a percentage. For example, where myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells are at a ratio of about 19:1 , this is equivalent to about 95% of total cells being myocytes and / or myocyte progenitor cells and 5% of total cells being adipocytes and / or adipose progenitor cells. In this context, total cells means all myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells in combination in the composition. It will be appreciated that in an embodiment where other cell types are present (such as fibroblasts) these are not included in calculating the percentage, or the ratio of the cells of (i) and (ii) in the composition. In some examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of at least about 13:1.

[0183] In another example, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of at least about 19:1 .

[0184] In another example, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of at least about 32:1 .

[0185] In another example, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a maximum ratio of about 49:1.

[0186] In some examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13: 1 to about 49: 1.

[0187] In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13: 1 to about 45: 1.

[0188] In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13: 1 to about 40: 1.

[0189] In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13: 1 to about 35: 1.

[0190] In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13: 1 to about 30: 1.

[0191] In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13: 1 to about 25: 1.

[0192] In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13:1 to about 19:1.

[0193] In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13:1 , about 14:1 , about 15:1 , about 16:1 , about 17:1 , about 18:1 , about 19:1 , or about 20:1. In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 21 :1 , about 22:1 , about 23:1 , about 24:1 , about 25:1 , about 26:1 , about 27:1 , about 28:1 , about 29:1 , or about 30:1. In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 31 :1 , about 32: 1 , about 33: 1 , about 34: 1 , about 35: 1 , about 36: 1 , about 37:1 , about 38:1 , about 39:1 , or about 40:1. In other examples, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 41 :1 , about 42: 1 , about 43: 1 , about 44: 1 , about 45: 1 , about 46: 1 , about 47:1 , about 48:1 , or about 49:1.

[0194] Suitably, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 13:1 (or 93:7).

[0195] Suitably, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 19:1 (or 95:5).

[0196] Suitably, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 32:1 (or 97:3).

[0197] Suitably, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 49:1 (or 98:2).

[0198] Suitably, the myocytes and / or myocyte progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio from about 93:7 to about 98:2 (for example, 93:7, 95:5, 97:3 or 98:2).

[0199] The present inventors have found that when a composition is provided with cells of (i) and (ii) at the above-mentioned ratios, overall cell proliferation is increased. This finding gives rise to a further aspect of the invention which provides a method for increasing cellular proliferation and / or differentiation, the method comprising the steps of:

[0200] (a) providing a composition, the composition comprising:

[0201] (i) myocytes and / or myocyte progenitor cells; and

[0202] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and

[0203] (b) co-culturing the cells in the composition to produce a cultivated animal biomass product.

[0204] In examples where the composition comprises:

[0205] (i) fibroblasts and / or fibroblast progenitor cells; and

[0206] (ii) adipocytes and / or adipose progenitor cells, the cells in the composition may be provided at a ratio from about 9:1 to about 99:1 of fibroblasts and / or fibroblast progenitor cells to adipocytes and / or adipose progenitor cells (i.e. cells of (i) to cells of (ii)). The term “ratio” refers to the number of fibroblasts and / or fibroblast progenitor cells to adipocytes and / or adipose progenitor cells. For example, a ratio of 9:1 means that for every 9 fibroblasts and / or fibroblast progenitor cells that are present in the composition, there is 1 adipocyte or adipose progenitor cell.

[0207] As it will be clear to a person of skill in the art, a ratio of 9:1 is equivalent to a ratio of 18:2, 27:3, 36:4, 45:5, and so on. Therefore, when fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells, are at a ratio of about 9:1 , about 18 fibroblasts and / or fibroblast progenitor cells will be present and about 2 adipocytes and / or adipose progenitor cells will be present, and so on. Likewise, if about 9x106fibroblasts and / or fibroblast progenitor cells are present, about 1 xio6adipocytes and / or adipose progenitor cells will be present in a ratio of 9:1.

[0208] A described above, ratio can be presented in the form of a percentage. For example, where fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells are at a ratio of about 9:1 , this is equivalent to about 90% of total cells being fibroblasts and / or fibroblast progenitor cells and 10% of total cells being adipocytes and / or adipose progenitor cells. In this context, total cells means all fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells in combination in the composition. It will be appreciated that in an embodiment where other cell types are present (such as myocytes) these are not included in calculating the percentage, or the ratio of the cells of (i) and (ii) in the composition.

[0209] In some examples, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of at least about 9:1.

[0210] In some examples, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 9: 1 to about 99: 1 .

[0211] In other examples, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 97:3 to about 93:7. These ratios are particularly useful for promoting proliferation.

[0212] In other examples, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 98:2 to about. 91 :9. These ratios are particularly useful for promoting tissue biomass (e.g. as represented by total collagen at day 7). In other examples, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 95:5 to about 9:1. These ratios are particularly useful for promoting tissue biomass (e.g. as represented by total protein at day 7).

[0213] In other examples, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 99:1 to about 92:8. These ratios are particularly useful for promoting tissue biomass (e.g. as represented by total protein at day 14).

[0214] In other examples, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio of about 95:5. This ratio is particularly useful for promoting tissue biomass (e.g. as represented by total collagen at day 14).

[0215] Suitably, the fibroblasts and / or fibroblast progenitor cells, and adipocytes and / or adipose progenitor cells may be in the composition at a ratio from about 9:1 to about 99:1 (for example, 9:1 ; 91 :9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:1).

[0216] The present inventors have found that when a composition is provided with cells of (i) and (ii) at the above-mentioned ratios, overall cell proliferation is increased. This finding gives rise to a further aspect of the invention which provides a method for increasing cellular proliferation and / or differentiation, the method comprising the steps of:

[0217] (a) providing a composition, the composition comprising:

[0218] (i) fibroblasts and / or fibroblast progenitor cells; and

[0219] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9:1 to about 99:1 ; and

[0220] (b) co-culturing the cells in the composition to produce a cultivated animal biomass product.

[0221] In the context of the methods of the invention, such as a method for cultivating animal biomass, or increasing cellular proliferation and / or differentiation, the composition may further comprise cell culture medium and / or a macromolecular crowding (MMC) agent. Examples of suitable cell culture media and MMC agents are provided elsewhere herein.

[0222] The method of cultivating animal biomass in vitro comprises a step of co-culturing the cells in the composition to produce a cultivated animal biomass product. The term “co-culturing” as used herein refers to culturing (i.e. keeping the cells in an artificial environment under conditions favouring growth, differentiation, and / or continued viability) of two or more different cell types. Culturing may be on a substrate or in suspension. As it will be appreciated by a person of skill in the art, whether culturing is on a substrate or in suspension may depend upon the type of cells being cultured. Suitably, when the cells are selected from the group consisting of fibroblasts, fibroblast progenitor cells, myocytes, myocyte progenitor cells, adipocytes, adipose progenitor cells, or a mixture of any two or more thereof, due to the adherent nature of these cells, culturing may be on a substrate. Suitable substrates are mentioned elsewhere herein. By contrast, mono-culture may be defined as a the culture in vitro of a population of cells of the same type, with type being defined as a specific set of morphological, phenotypical, and functional characteristics.

[0223] In the context of the present disclosure, the two or more different cell types comprise at least:

[0224] (a1) (i) myocytes and / or myocyte progenitor cells, and (ii) adipocytes and / or adipose progenitor cells or

[0225] (a2) (i) fibroblasts and / or fibroblast progenitor cells, and (ii) adipocytes and / or adipose progenitor cells.

[0226] When co-culture involves the step of differentiating, this may involve differentiating adipose progenitor cells and / or myocyte progenitor cells and / or fibroblast progenitor cells (as appropriate). Suitably, adipose progenitor cells may be differentiated into adipocytes. Suitably, myocyte progenitor cells may be differentiated into myoblasts and / or myocytes. Suitably, myoblasts may be differentiated into myocytes. Suitably, fibroblast progenitor cells may be differentiated into fibroblasts.

[0227] When two or more different cell types are differentiated, the step of differentiation of the different cell types may occur simultaneously or sequentially. As it will be appreciated, differentiation may occur when cells that have the potential to differentiate are cultured in the presence of appropriate cell differentiation factors. Methods for differentiating cells such as adipose progenitor cells, fibroblast progenitor cells and myocyte progenitor cells will be known to those skilled in the art.

[0228] Suitably, the co-cultured cells may be cultured as a homogeneous mixture, i.e.

[0229] (a1) the (i) myocytes and / or myocyte progenitor cells, and (ii) adipocytes and / or adipose progenitor cells are mixed with one another and randomly dispersed; or

[0230] (a2) the (i) fibroblasts and / or fibroblast progenitor cells, and (ii) adipocytes and / or adipose progenitor cells are mixed with one another and randomly dispersed. Alternatively or additionally, the co-cultured cells may be cultured such that the cells of (i) and (ii) are not mixed with one another, or at least the majority of the cells are not mixed with one another. For example, the cells of (i) and (ii) may form distinctive layers or be present in distinctive zones on a substrate. In such an embodiment, it will be appreciated that at least a proportion of the cells of (i) may be in physical contact with at least a proportion of cells of (ii). In another embodiment, the two cell types may be physically separated but communicating biochemically. Suitably, the two cell types may be physically separated but communicating biochemically when they are in fluid communication. The fluid may be cell culture medium.

[0231] The terms "cell culture medium" and "culture medium" (plural "media" in each case) refer to a nutritive solution for cultivating live cells and may be used interchangeably. As mentioned, the medium may be supplemented with differentiation factors. Such a medium may be referred to as a “differentiation medium”. However, it will be appreciated that a differentiation medium is a type of cell culture medium.

[0232] The cell culture medium may be a complete formulation, i.e. , a cell culture medium that requires no supplementation to culture cells, or may be an incomplete formulation, i.e., a cell culture medium that requires supplementation or may be a medium that may supplement an incomplete formulation or in the case of a complete formulation, may improve culture or culture results.

[0233] Various cell culture media will be known to those skilled in the art, who will also appreciate that the type of cells to be cultured may dictate the type of culture medium to be used.

[0234] Merely by way of example and not limitation, the culture medium may be selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 (F-12), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI-1640, Ham's F-10, aMinimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium(IMDM), or any combination thereof. Other media that are commercially available (e.g., from Thermo Fisher Scientific, Waltham, MA) or that are otherwise known in the art can be equivalently used in the context of this disclosure. Again, only by way of example, the media may be selected from the group consisting of 293 SFM, CD-CHO medium, VP SFM, BGJb medium, Brinster's BMOC-3 medium, cell culture freezing medium, CMRL media, EHAA medium, eRDF medium, Fischer's medium, Gamborg's B-5 medium, GLUTAMAX™ supplemented media, Grace's insect cell media, HEPES buffered media, Richter's modified MEM, IPL-41 insect cell medium, Leibovitz's L-15 media, McCoy's 5A media, MCDB 131 medium, Media 199, Modified Eagle's Medium (MEM), Medium NCTC-109, Schneider's Drosophila medium, TC-100 insect medium, Waymouth's MB 752 / 1 media, William's Media E, protein free hybridoma medium II (PFHM II), AIM V media, Keratinocyte SFM, defined Keratinocyte SFM, STEMPRO® SFM, STEMPRO® complete methylcellulose medium, HepatoZYME-SFM, Neurobasal™ medium, Neurobasal-A medium, Hibernate™ A medium, Hibernate E medium, Endothelial SFM, Human Endothelial SFM, Hybridoma SFM, PFHM II, Sf 900 medium, Sf 900 II SFM, EXPRESS FIVE® medium, CHO-S-SFM, AMINOMAX-II complete medium, AMINOMAX-C100 complete medium, AMINOMAX-C140 basal medium, PUB-MAX™ karyotyping medium, KARYOMAX bone marrow karyotyping medium, and KNOCKOUT D-MEM, or any combination thereof. Suitably, the combination may be of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 (F-12) (referred to in the Examples section of the present disclosure as DMEM / F12.

[0235] The medium may be liquid, semi-solid, or solid growth medium.

[0236] In some embodiments, the cell culture medium may comprise serum. In other embodiments, the culture medium may comprise low levels of serum. Such a medium may be referred to as cell culture medium with “reduced serum”. The term “reduced serum” is defined herein below. In other embodiments, the culture medium may be serum-free. When the cell culture medium has reduced serum, it may be said that the co-culture is performed in reduced-serum conditions. By the same token, when the cell culture medium is serum-free, it may be said that the co-culture is performed in serum-free conditions.

[0237] The term “reduced-serum” conditions as used herein is used to describe the presence of serum in the co-culture, but at a lower level than would usually be used for optimal co-culture of the cells of interest. For example, it is accepted in the field that muscle cells (such as myocytes and / or myocyte progenitor cells) or fat cells (such as adipocytes and / or adipose progenitor cells) or skin cells (such as fibroblasts and / or fibroblast progenitor cells) are typically cultured in a cell culture medium that comprises at least 10% (v / v) serum for cell growth, and at least 5% (v / v) serum for cell differentiation. Thus, in the context of the present disclosure, a co-culture medium that has reduced-serum may have no more than about 2% (v / v) serum may therefore be considered “reduced-serum” conditions. Suitably, in the present invention, co-culturing may be performed in serum-free or reduced-serum conditions, wherein the reduced-serum conditions comprise no more than about 2% (v / v) serum in the cell culture medium, no more than about 1.5% (v / v) serum in the cell culture medium, no more than about 1% (v / v) serum in the cell culture medium, no more than about 0.5% (v / v) serum in the cell culture medium, or less. Reduced-serum conditions may have (substantially) no serum. In this context, “substantially no serum” means that there are no more than trace amounts of serum. Trace amounts may be defined as a maximum of 0.1 % (v / v) serum. For example, the reduced-serum cell culture medium may have about 0.1% to about 1.5% (v / v) serum. For example, the reduced-serum cell culture medium may have about 0.1 % to about 1% (v / v) serum. As another example, the reduced-serum cell culture medium may have about 0.1% to about 0.5% (v / v) serum.

[0238] Cell culture media wherein there is no detectable serum are referred to herein as “serum-free” cell culture media (SFM). Typically, for media that contains serum, the serum is added as a supplement at the start of, or during cell culture. The term “serum-free” cell culture medium therefore includes cell culture media which have not been supplemented with serum. The term “serum-free” is very well known in the art.

[0239] As would be clear to a person of skill in the art, “serum-free” media may comprise a number of additives and supplements, provided that it does not contain detectable levels of serum.

[0240] The serum-free or reduced-serum cell culture media described herein are particularly advantageous as they provide a more chemically defined media for cell culture, using reagents that are more sustainable, and with a lower risk of contamination compared to equivalents that are reliant on serum. Furthermore, in the context of the present discourse which may include the production of an edible, the use of serum-free or reduced-serum cell culture media may be advantageous due to reduced risk of pathogen contamination which is typically associated with the use of serum.

[0241] Suitably, in the step of culturing the cells in the composition to produce a cultivated animal biomass product, the composition may comprise a macromolecular crowding (MMC) agent. Therefore, in the context of the method for cultivating animal biomass in vitro, it can be said that the step of co-culturing is performed in the presence of an MMC agent.

[0242] Macromolecular crowding (MMC) is a biophysical phenomenon based on the principles of excluded-volume effect. It involves the addition of macromolecules to culture media. Following the principles of excluded volume effect (two molecules cannot occupy the same space at the same time), MMC significantly increases rates and kinetics of biochemical reactions and biological processes. According to the excluded volume effect theory, the volume of a solution that is excluded to a particular molecule is dependent on the sum of nonspecific hindrances (governed by size and shape) and electrostatic repulsions (governed by electrical charge) between the background molecules. Crowding is a result of the reduction of the available solvent volume by a macromolecule, which can be mobile or fixed. Crowding hinders solute diffusion, thereby increasing the effective solute concentration. This, in turn, increases the chemical potential of the solute. Crowding can therefore shift reaction equilibria and change the rates of chemical reactions. Crowding has therefore been used extensively to study polymer looping dynamic properties, DNA structure, condensation, replication, and stability, for example. Macromolecular crowding influences many critical processes including cell adhesion, migration, proliferation as well as extracellular matrix formation and remodelling. These effects have been shown herein to positively affect cell culture of fat (such as adipocytes and / or adipose progenitor cells), muscle cells (such as myocytes and / or myocyte progenitor cells, or differentiated forms thereof of such as myocytes), skin cells (such as fibroblasts and / or fibroblast progenitor cells) or indeed combinations thereof.

[0243] Several MMC agents are known. In the context of the cell culture media, methods and compositions described herein, the MMC agent may be one or more MMC agents selected from the group consisting of: PVP, PEG8, PEG35, PVP40, Carrageenan, Ficoll® 70, and Ficoll® 400; or a combination thereof. Suitably, the MMC agent may be one or more MMC agents selected from the group consisting of: PVP, PEG8, PEG35, PVP40, Ficoll® 70, and Ficoll® 400; or a combination thereof.

[0244] Suitably, the step of co-culturing may be performed in serum-free media comprising a MMC agent. Suitably, the MMC agent may be selected from the group consisting of: PVP, PEG8, PEG35, PVP40, Carrageenan, Ficoll® 70, and Ficoll® 400; or a combination thereof. Suitable, in the methods of the present discourse, the step of co-culturing may be performed in serum-free media comprising a MMC agent selected from the group consisting of: PVP, PEG8, PEG35, PVP40; or a combination thereof.

[0245] Suitably, the step of co-culturing may be performed in serum reduced media comprising a MMC agent. Suitably, the MMC agent may be selected from the group consisting of: PVP, PEG8, PEG35, PVP40, Carrageenan, Ficoll® 70, and Ficoll® 400; or a combination thereof. Suitable, in the methods of the present discourse, the step of co-culturing may be performed in serum reduced media comprising a MMC agent selected from the group consisting of: PVP, PEG8, PEG35, PVP40; or a combination thereof.

[0246] Suitably, the MMC agent may be PVP360. Suitably, PVP360 may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 10 g / L to about 30g / L. More suitably, the step of co-culturing may be performed in serum-free media comprising PVP360 at a concentration of from about 10g / L to about 20g / L. For example, the step of co-culturing may be performed in serum-free media comprising PVP360 at a concentration of from about 10g / L to about 20g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum-free media comprising PVP360 at a concentration of from about 10g / L to about 20g / L, wherein the co-culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum-free media comprising PVP360 at a concentration of about 10g / L to about 20g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12.

[0247] Suitably, the MMC agent may be PVP360 (also referred to as “PVP” in the examples and figures below). Suitably, PVP360 may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 10 g / L to about 30g / L. More suitably, the step of co-culturing may be performed in reduced-serum media comprising PVP360 at a concentration of from about 10g / L to about 20g / L. For example, the step of co-culturing may be performed in reduced-serum media comprising PVP360 at a concentration of from about 10g / L to about 20g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced-serum media comprising PVP360 at a concentration of from about 10g / L to about 20g / L, wherein the co- culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced- serum media comprising PVP360 at a concentration of about 10g / L to about 20g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12.

[0248] Suitably, the MMC agent may be PEG8. Suitably, PEG8, may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 5 g / L to about 15g / L. More suitably, the step of co-culturing may be performed in serum-free media comprising PEG8 at a concentration of from about 7g / L to about 9g / L, for example 8.25g / L. For example, the step of co-culturing may be performed in serum-free media comprising PEG8 at a concentration of about 8.25g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum- free media comprising PEG8 at a concentration of about 8.25g / L, wherein the co-culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum-free media comprising PEG8 at a concentration of about 8.25g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12.

[0249] Suitably, the MMC agent may be PEG8. Suitably, PEG8, may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 5 g / L to about 15g / L. More suitably, the step of co-culturing may be performed in reduced-serum media comprising PEG8 at a concentration of from about 7g / L to about 9g / L, for example 8.25g / L. For example, the step of co-culturing may be performed in reduced-serum media comprising PEG8 at a concentration of about 8.25g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced-serum media comprising PEG8 at a concentration of about 8.25g / L, wherein the co- culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced- serum media comprising PEG8 at a concentration of about 8.25g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12.

[0250] Suitably, the MMC agent may be PEG35. Suitably, PEG35 may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 10 g / L to about 30g / L. More suitably, the step of co-culturing may be performed in serum-free media comprising PEG8 at a concentration of from about 20g / L. For example, the step of co-culturing may be performed in serum-free media comprising PEG35 at a concentration of about 20g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum-free media comprising PEG35 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum-free media comprising PEG35 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12.

[0251] Suitably, the MMC agent may be PEG35. Suitably, PEG35 may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 10 g / L to about 30g / L. More suitably, the step of co-culturing may be performed in reduced-serum media comprising PEG8 at a concentration of from about 20g / L. For example, the step of co-culturing may be performed in reduced-serum media comprising PEG35 at a concentration of about 20g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced-serum media comprising PEG35 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced-serum media comprising PEG35 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12.

[0252] Suitably, the MMC agent may be PVP40. Suitably, PVP40 may be at a concentration of from about 10 g / L to about 70 g / L, or for example from about 20 g / L to about 50g / L, or for example from about 20 g / L to about 40g / L. More suitably, the step of co-culturing may be performed in serum-free media comprising PVP40 at a concentration of from about 30g / L. For example, the step of co-culturing may be performed in serum-free media comprising PVP40 at a concentration of about 30g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum-free media comprising PVP40 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in serum- free media comprising PVP40 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12. Suitably, the MMC agent may be PVP40. Suitably, PVP40 may be at a concentration of from about 10 g / L to about 70 g / L, or for example from about 20 g / L to about 50g / L, or for example from about 20 g / L to about 40g / L. More suitably, the step of co-culturing may be performed in reduced-serum media comprising PVP40 at a concentration of from about 30g / L. For example, the step of co-culturing may be performed in reduced-serum media comprising PVP40 at a concentration of about 30g / L, wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced-serum media comprising PVP40 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 72 hours, optionally wherein the media is DMEM / F12. In another example the step of co-culturing may be performed in reduced-serum media comprising PVP40 at a concentration of about 20g / L, wherein the co-culturing step described herein may be performed for at least 120 hours, wherein the media is DMEM / F12.

[0253] Polyvinylpyrrolidone 360 kDa (referred to herein as PVP or PVP360) used as a macromolecular crowder together with serum has been shown to increase human dermal fibroblast cell proliferation and extracellular matrix deposition as indicated by increases in collagen type I production. The molecular structure of PVP360 is also well known. See for example Kariduraganavar et al., Natural and Synthetic Biomedical polymers; chapter 1 , 2014, pages 1 to 31.

[0254] Polyethylene glycol (PEG) is a commonly used macromolecular crowder having effects on in vitro experiments, such as influencing ligand affinity and rate of enzymatic reaction and promoting extracellular matrix deposition when used with serum. Polyethylene glycol is prepared by polymerization of ethylene oxide and is commercially available over a wide range of molecular weights, from 300 Da to 10,000 kDa (Alister et al., Angewandte Chemie International Edition Volume 48, Issue 7 p. 1248-1252). For example, Polyethylene Glycol 8 kDa (PEG8) is a non-toxic polyether with hydrophilic head allowing to dilution in aqueous solutions. Macromolecular crowding induced by PEG8 can modulate reactions by increasing substrate binding at high concentrations as well as increasing proliferation bacterial strains. The molecular structure of PEG8 is well known, see for example Sigma-Aldrich, Cas Number 25322-68-3, linear formula: H(OCH2CH2)nOH and Hyun-Jun Jang et al., Toxicol Res. 2015 Jun; 31 (2): 105-136.

[0255] An alternative PEG that may be used herein is PEG35. The molecular structure of PEG35 is also well known, see for example Hyun-Jun Jang et al., Toxicol Res. 2015 Jun; 31 (2): 105-136.

[0256] Polyvinylpyrrolidone 40 kDa (PVP40) is a water-soluble polymer with a variety of uses including in beverage stabilization and medical uses where it is used as plasma volume expander. PVP40 in combination with serum has also been shown to be an effective macromolecular crowder with treatment increasing both collagen type I and proliferation of human dermal fibroblasts. The molecular structure of PVP40 is well known, see for example Sigma Aldrich, Cas Number 9003- 39-8, linear formula (C6H9NO)n. and Kariduraganavar et al., Natural and Synthetic Biomedical polymers; chapter 1 , 2014, pages 1 to 31.

[0257] Carrageenans (also known as carrageenins) are a family of natural linear sulphated polysaccharides that are extracted from red edible seaweeds. The most well-known and still most important red seaweed used for manufacturing the hydrophilic colloids to produce carrageenan is Chondrus crispus (Irish moss) which is a dark red parsley-like plant that grows attached to the rocks. Carrageenans are widely used in the food industry, for their gelling, thickening, and stabilizing properties. Their main application is in dairy and meat products, due to their strong binding to food proteins.

[0258] All carrageenans are high-molecular-weight polysaccharides and mainly made up of alternating 3-linked b-D-galac-topyranose (G-units) and 4-linked a-D-galactopyranose (D-units) or 4-linked 3,6-anhydro-a-D-galactopyranose (DA-units), forming the disaccharide repeating unit of carrageenans. There are three main commercial classes of carrageenan: Kappa carrageenan, lota carrageenan and Lambda carrageenan. The molecular structures of different types of carrageenan are well known, see for example Hilliou, Adv Food Nutr Res. 2014;72:17-43.

[0259] Suitable the carrageenan may be lambda carrageenan. Carrageenans encompass a family of sulphated galactans originally extracted from red seaweed, where they have been found to play key structural functions. Traditionally, carrageenans are produced and used as crude extracts comprising different combinations of three molecular species defined by their sulphation and the presence or absence of anhygalactose. The lambda-carrageenan contains about of 35% ester sulfate and no anhygalactose, making it highly soluble in water and unable to form gels. In contrast, the iota-carrageenan and kappa-carrageenan contain less ester sulfate and about 30- 35% of 3,6-anhydrogalactose, making them insoluble in cold water and forming thermo-reversible gels in hot aqueous solutions. The different physicochemical and biological properties of lambda- carrageenan demonstrates that this molecular species is altogether distinct from the iota and kappa species, as well as from crude carrageenan extracts.

[0260] Carrageenan has been proposed to be a promising macromolecular crowder (MMC) for tissue engineering due to its ability to increase extracellular matrix production. Treatment of adipose- derived stem cells with carrageenan and serum has been shown to enhance extracellular matrix deposition of collagen type I, II and V, to increase cell proliferation as well as increasing osteogenesis, chondrogenesis and decreasing adipogenesis. Ficoll® 70 (also known as Poly(sucrose-co-epichlorhydrin)) is used as a macromolecular crowding agent in studies of cell volume signaling and protein refolding. It may be used in tissue engineering and macromolecular conformation research for the development, evaluation and use of macromolecular crowding (MMC) systems and configurations. The molecular structure of Ficoll® 70 is well known, see for example Sigma Aldrich, Cas Number 72146-89-5, and CN102690364A.

[0261] Ficoll® 400 (also known as Polysucrose 400) is a non-ionic synthetic polymer of sucrose used for cell separation and organ isolation. The molecular structure of Ficoll® 400 is well known, see for example Sigma Aldrich, Cas Number 26873-85-8, and CN102690364A and https: / / pubchem.ncbi.nlm.nih.gov / compound / Ficoll-400.

[0262] The MMC agents described herein may be used as a single supplement (wherein only one MMC agent is added to the cell culture medium), or they may be used in combination. Suitable combinations may be identified by a person of skill in the art. For example, a combination of at least two MMC agents may be used. Alternatively, a combination of at least three or at least four MMC agents may be used. The MMC agents may be used at any appropriate concentration within the cell culture media described herein.

[0263] Suitable final concentrations of MMC agents that are used in combination may be determined based on the disclosure provided herein, using routine methods known in the art.

[0264] Suitably, the composition and / or cell culture medium utilised in the methods of the present disclosure may comprise a further component selected from the group consisting of L-glutamine, L-alanyl-L-glutamine dipeptide, a natural and / or synthetic peptide growth factor (such as insulin, an insulin growth factor, a fibroblast growth factor, a transforming growth factor, a hepatocyte growth factor, a platelet-derived growth factors, and / or an epithelial growth factor), a neuregulin, a morphogenic protein, a vitamin (for example ascorbic acid, a\\-trans retinoic acid, thiamine, calciferol, tocopherol, riboflavin, niacin, biotin, pantothenic acid, folic acid, cobalamin, pyridoxine, and / or phytonadione), a carrier molecule (for example albumin, fetuin, and / or transferrin), an amino alcohol (such as ethanolamine, carbinoxamine, or chlorphenoxamine), a trace metal (such as selenium, zinc, aluminium, manganese, molybdenum, and / or iron) and a lipid (such as cholesterol, linoleic, stearic, oleic, palmitic, and / or uric acid), or analogues thereof, or activators or inhibitors of their molecular pathways.

[0265] Suitably, the composition and / or cell culture medium utilised in the methods of the present disclosure (for example in the context of co-culturing) may comprise L-alanyl-L-glutamine dipeptide, L-ascorbic acid 2-phosphate, and Insulin, Transferrin, Selenium, and Ethanolamine (ITS-X). Suitably such a cell culture medium may be serum-free DMEM / F12, and / or comprise a MMC agent as described herein above.

[0266] Suitably, the composition and / or cell culture medium utilised in the methods of the present disclosure may comprise L-alanyl-L-glutamine dipeptide, ascorbic acid 2-phosphate, sesquimagnesium salt hydrate, Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) and penicillin / streptomycin. Suitably such a cell culture medium may be serum-free DMEM / F12, and / or comprise a MMC agent as described herein above. For example, the MMC may be 8.25 g / L polyethylene glycol MW 8,000 (+PEG8); 20 g / L polyethylene glycol MW 35,000 (+PEG35); 30 g / L polyvinylpyrrolidone MW 40,000 (+PVP40); or 10 g / L polyvinylpyrrolidone MW 360,000 (+PVP360).

[0267] Suitably, the composition and / or cell culture medium utilised in the methods of the present disclosure may comprise 5mM L-alanyl-L-glutamine dipeptide, 1 mM ascorbic acid 2-phosphate, sesquimagnesium salt hydrate, 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) and 1% penicillin / streptomycin. Suitably such a cell culture medium may be serum-free DMEM / F12, and / or comprise a MMC agent as described herein above. For example, the MMC may be 8.25 g / L polyethylene glycol MW 8,000 (+PEG8); 20 g / L polyethylene glycol MW 35,000 (+PEG35); 30 g / L polyvinylpyrrolidone MW 40,000 (+PVP40); or 10 g / L polyvinylpyrrolidone MW 360,000 (+PVP360).

[0268] Suitably, the composition and / or cell culture medium utilised in the methods of the present disclosure (for example in the context of co-culturing) may comprise 4.5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate, and 1 % Insulin, Transferrin, Selenium, Ethanolamine (ITS-X) supplement. Suitably such a cell culture medium may be serum free, and / or comprise a MMC agent as described herein above.

[0269] Suitably, in one example, the composition comprises of cells, wherein the cells comprise or consist of (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells. In an embodiment, where the cells comprise of (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells, the (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells may account for at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or more of the cells. Suitably, the (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells may account for at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cell in the composition.

[0270] Suitably, in one example, the composition comprises of cells, wherein the cells comprise or consist of (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells. In an embodiment, where the cells comprise of (i) fibroblast and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells, the (i) fibroblast and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells may account for at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or more of the cells. Suitably, the (i) fibroblast and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells may account for at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cell in the composition.

[0271] In the context of the present disclosure, the term “providing” may comprise “seeding” of the cells in the composition.

[0272] Accordingly, in some embodiments the cells in the composition (e.g. the (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells) may be seeded at a ratio of from about 13:1 to about 49:1. Other suitable ratios are discussed elsewhere herein.

[0273] Accordingly, in other embodiments the cells in the composition (e.g. the (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells) may be seeded at a ratio of from about 9:1 to about 99:1. Other suitable ratios are discussed elsewhere herein.

[0274] The term “seeding” as used herein refers to placing cells on a substrate or into a cell culture medium. Adherent cells may be placed on a substrate by placing them in an aqueous solution (such as a cell culture medium) and allowing them to attach to the substrate. Suspension cells may be placed directly in an aqueous solution (such as a cell culture medium).

[0275] Suitably, the cells of (i) and (ii) may be seeded on a substrate at a cell density of from about 1 ,000 cells / cm2to about 200,000,000 cells / cm2, from about 5,000 cells / cm2to about 100,000,000 cells / cm2, or from about 15,000 cells / cm2to about 50,000,000 cells / cm2. Suitably, the cells may be seeded at a cell density from about 10,000 cells / cm2to about 200,000 cells / cm2. For clarity, when cells are seeded at a cell density of about 200,000 cells / cm2, it means that 200,000 cells are seeded per cm2of substrate area. It is common knowledge in the art that cell seeding density is important for cell proliferation, differentiation, and extracellular matrix (ECM) synthesis. In the context of the present invention, a cell seeding density of at least 10,000 cells / cm2has been observed to be particularly useful for animal biomass cultivation. It will be appreciated these seeding densities which are represented as cell number per cm2are particularly relevant in the context of adherent cells.

[0276] Suitably, the cells of (i) and (ii) may be seeded on a substrate at a cell density of from about 5 cells / cm3to about 5x108cells / cm3, from about to about 10 cells / cm3to about 1 x108cells / cm3, or from about 20 cells / cm3to about 5x107cells / cm3. Suitably, the cells may be seeded at a cell density of at least about 5 cells / cm3. Suitably, the cells may be seeded at a cell density of about 5 cells / cm3. For clarity, when cells are seeded at a cell density of about 5 cells / cm3, it means that 5 cells are seeded per cm3of an aqueous solution (such as cell culture media). It will be appreciated these seeding densities which are represented as cell number per cm3are particularly relevant in the context of non-adherent cells (i.e. cells that grow in suspension).

[0277] The cells of (i) and (ii) may be seeded simultaneously or sequentially. When seeded simultaneously the cells may be placed into a cell culture medium as a mixture, allowing both cells of (i) and (ii) to attach to the substrate substantially simultaneously. Alternatively, one of the cell types may be placed into the cell culture medium first, followed by second type. The timing between placing the first and second cell types into the cell culture medium would not be sufficient to allow the first cell type to attach to the substrate, thereby allowing both cell types to attach to the substrate substantially simultaneously. When seeded sequentially, the first cell type placed into the cell culture medium would be allowed to substantially attach to the substrate, prior to placing the second cell type into the cell culture medium. In some embodiments, the cells may be seeded on different parts of the substrate.

[0278] In some examples, the cell seeding density may be made up from two or more cell types at a particular ratio. By way of an example, the cells of (i) and (ii) may be seeded as a composition comprising of a ratio of 19:1 and at a density of about 10,000 cells / cm2. In such an example the seeding density of (i) will be about 9.5x103cells / cm2and seeding density of (ii) will be about 0.5x103cells / cm2. Alternatively, cells of (i) may be seeded at a cell density of 9.5x103cells / cm2, followed by seeding of cells of (ii) at a density of 0.5x103cells / cm2.

[0279] In a suitable embodiment, the cells of each of the two cell types need not to be seeded at a single time point. Instead, for example, a first proportion of cells of (i) and / or cells of (ii) may be seeded at an initial time point, and a second proportion of cells of (i) and / or cells of (ii) may be seeded at a later time point. By way of example, 9,500 cells / cm2may be seeded on day 1 , and then 500 cells / cm2may be seeded on day 2 to give a final cell seeding density of 10,000 cells / cm2, or in other words, only 10,000 cells / cm2would have been seeded.

[0280] The term “substrate” as used herein refers to a material or substance which has a surface for cell adherence. The substrate may be cellularised (i.e. comprising cells) or may be cell free. The surface may be the outside part or uppermost layer of a material or substance. The substrate may be porous or dynamic, which permits adherence within the substrate. The substrate can be 2- dimensional (2D) or 3-dimensional (3D). An example of a 2D substrate is the surface of a culture vessel, such as a tube, a flask, a dish or a plate comprising a plurality of wells. The culture vessel may be a glass, plastic, or metal container that can provide an aseptic environment for culturing cells. An example of a 3D surface is a scaffold, such as a gel scaffold (e.g. hydrogel) or extracellular matrix (ECM). Suitably, the ECM may be decellularized.

[0281] Examples of cellular substrates are discussed elsewhere herein.

[0282] The co-culturing step described herein may be performed from about 6 hours to about 336 hours, or from about 12 hours to about 120 hours, or from about 24 hours to about 48 hours.

[0283] Suitably, the co-culturing step described herein may be performed for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours.

[0284] Suitably, the co-culturing step described herein may be performed for at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 168 hours, at least 192 hours, at least at least 216 hours, at least 240 hours, at least 264 hours, at least 288 hours, at least 312 hours, or at least 336 hours. More suitably, the co-culturing step described herein may be performed for at least 120 hours.

[0285] Suitably, the co-culture step lasts until an animal biomass product is obtained.

[0286] In another aspect, the present invention provides a composition for cultivating animal biomass. Suitably, the composition is for cultivating animal biomass in vitro.

[0287] Suitably, the composition may comprise (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, wherein the ratio is from about 13:1 to about 49:1 (for example from about 19:1 to about 33:1).

[0288] In an alternative example, the composition may comprise (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, wherein the ratio is from about 9:1 to about 99:1.

[0289] Suitable embodiments of the composition are described hereinabove in the context of the methods of the invention. For example, suitable ratios of cells of (i) and (ii) that are described in the context of the methods of the invention may also apply equally to the composition of the invention. Examples of cell culture media and / or MMC agents described in the context of the methods of the invention may also apply equally to the composition of the invention.

[0290] Suitably, the composition may comprise cell culture medium. Suitably, the composition is a serum- free or reduced-serum composition.

[0291] Suitably, the composition may comprise a serum-free media and a MMC agent. Suitably, the MMC agent may be selected from the group consisting of: PVP, PEG8, PEG35, PVP40, Carrageenan, Ficoll® 70, and Ficoll® 400; or a combination thereof. Suitable, the MMC agent may be selected from the group consisting of: PVP, PEG8, PEG35, PVP40; or a combination thereof.

[0292] Suitably, the MMC agent may be PVP. Suitably, PVP360 may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 10 g / L to about 30g / L. More suitably, the composition may comprise serum-free media and PVP360 at a concentration of from about 10g / L to about 20g / L. For example, the composition may comprise serum-free media and PVP360 at a concentration of from about 10g / L to about 20g / L, wherein the media is DMEM / F12.

[0293] Suitably, the MMC agent may be PEG8. Suitably, PEG8, may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 5 g / L to about 15g / L. More suitably, PEG8 may be at a concentration of from about 7g / L to about 9g / L, for example 8.25g / L. For example, the composition may comprise serum-free media and PEG8 at a concentration of about 8.25g / L, wherein the media is DMEM / F12.

[0294] Suitably, the MMC agent may be PEG35. Suitably, PEG35 may be at a concentration of from about 5 g / L to about 50 g / L, or for example from about 10 g / L to about 30g / L. More suitably, the composition may comprise serum-free media and PEG35 at a concentration of from about 20g / L. For example, the composition may comprise serum-free media and PEG35 at a concentration of about 20g / L, wherein the media is DMEM / F12.

[0295] Suitably, the MMC agent may be PVP40. Suitably, PVP40 may be at a concentration of from about 10 g / L to about 70 g / L, or for example from about 20 g / L to about 50g / L, or for example from about 20 g / L to about 40g / L. More suitably, the composition may comprise serum-free media and PVP40 at a concentration of from about 30g / L. For example, the composition may comprise serum-free media and PVP40 at a concentration of about 30g / L, wherein the media is DMEM / F12.

[0296] In a further aspect, the present invention provides a cultivated animal biomass product produced by the method of the invention.

[0297] In a further aspect, the present invention provides a method for cultivating meat in vitro, the method comprising:

[0298] (a) providing a composition, the composition comprising:

[0299] (i) myocytes and / or myocyte progenitor cells; and

[0300] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0301] (c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product.

[0302] Suitably, the cellular substrate may further comprise adipocytes and / or adipose progenitor cells. Optionally, the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells may be present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9: 1 to about 99: 1 .

[0303] This aspect of the invention is based on the inventors surprising finding that when a composition comprising: (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49:1 are contacted with the cellular substrate, and co-cultured, the dry and / or wet weight of the cultivated meat product is increased, even when the culture conditions are serum free. Without wishing to be bound by this hypothesis, the present inventors believe that that the presence of a cellular substrate can therefore provide beneficial effects to the cells of (i) and (ii), which typically would be expected to be provided by the presence of serum.

[0304] The term “contacting” as used herein refers to bring the composition sufficiently close in proximity to the cellular substrate comprising fibroblasts and / or fibroblast progenitor cells (optionally together with adipocytes and / or adipose progenitor cells) to allow the composition and cellular composite to react or interact. Suitably, contacting may mean physically contacting some or all of the composition (for example some or all of the cells in the composition) with some or all of the cellular substrate.

[0305] The term “cellular substrate” refers to a surface comprising cells which may be used to grow the cells of the composition on. According, the cells of the composition may adhere to the cellular substrate. It will be appreciated that the cells of the composition may adhere to the cellular substrate by being seeded on the cellular substrate.

[0306] The cellular substrate may itself be adhered to a different substrate (such as cellular substrates of the same or similar type, cellular substrates of a different type, or a non-cellular substrate). Examples of “substrates” are disclosed elsewhere herein. The cellular substrate may be 2D or 3D.

[0307] Alternatively, the cellular substrate may not be adhered to a substrate itself. In such an embodiment the cells of the composition may be in contact with a substrate (such as a cell culture vessel), and the cellular substrate may be overlayed on top of the cells of the composition). Such an embodiment may give rise to a cellular composite as described elsewhere herein.

[0308] Suitably, the cellular substrate comprises fibroblasts and / or fibroblast progenitor cells (optionally together with adipocytes and / or adipose progenitor cells).

[0309] The term “fibroblasts” as used herein refers to a cell that in vivo contributes to the formation of connective tissue, a fibrous cellular material that supports and connects other tissues or organs in the body. The fibroblast may secrete collagens and other fibrillar proteins that help maintain the structural framework of tissue. In the context of the present discourse, the tissue may be the cultivated animal biomass product. The term fibroblast is well known in the art, and the skilled person will be well aware of methods to determine whether a cell is a fibroblast or not. Merely by way of example, fibroblasts may be identified by positive expression of the mesenchymal markers vimentin and PDGFR alpha, or by microscopic analysis due to their characteristic morphology.

[0310] Fibroblast cells may produce an ECM. The presence of the ECM may aid in the cultivation of a more animal-like cultivated animal biomass product. In the context of the present disclosure, the ECM is not classified as a scaffold, as it is natively produced by the cells of the cultivated animal biomass. Accordingly, even when the ECM is present in the cultivated animal biomass described herein, it may be said the cultivated animal biomass is scaffold-free, or free of an exogenous scaffold.

[0311] The term “fibroblast progenitor cells” as used herein refers to cells that have the potential to differentiate into fibroblasts. Suitably, the fibroblast progenitor cells may be mesenchymal stem cells.

[0312] It will be appreciated that the fibroblasts and / or fibroblast progenitor cells may be animal cells as described elsewhere herein. Accordingly, suitably, the fibroblasts and / or fibroblast progenitor cells are not human cells. Suitably, the fibroblasts and / or fibroblast progenitor cells are porcine. More suitably, the cellular substrate comprises or consists of fibroblasts (for example porcine fibroblasts).

[0313] Suitably, the cellular substrate comprising fibroblasts and / or fibroblast progenitor cells may comprise a layer of cells. Suitably, the layer may be continuous or discontinuous. A continuous layer is a sheet-like structure of cells. A continuous layer may a confluent layer. A discontinuous layer refers to a porous sheet-like structure of cells. A discontinuous layer may be referred to as a non-confluent layer. Suitably, the layer may be a monolayer or a multilayer. Suitably, the fibroblasts may aid in the differentiation of other cells. More suitably, the fibroblasts may aid in the differentiation of myocytes or myocyte progenitor cells to differentiate and form muscle fibres. Said muscle fibres may be integrated with fat. Therefore, fibroblasts may aid formation of animal tissue. The present inventors believe that fibroblasts (for example a confluent layer of fibroblasts) may provide a rich extracellular matrix environment, along with cell signalling cues, for the myocyte progenitor cells (such as muscle satellite cells) to differentiate and form myotubular tissue with integrated fat, allowing high quality, high biomass tissue to be produced.

[0314] Suitably the cells the composition may be seeded onto the cellular substrate. However, in an embodiment where the cellular substrate is a discontinuous layer, a proportion of the cells of the composition may not be seeded into the cellular substrate. Instead, in such an embodiment, said proportion may be seeded on a substrate.

[0315] In the context of the method of cultivating meat in vitro, wherein the method comprises the step of contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product, it will be appreciated that the term “co-culturing” refers to culturing (i.e. keeping the cells in an artificial environment under conditions favouring growth, differentiation, and / or continued viability) at least three different cell types. The three cell types in this context are the two cell types of the composition , as well as fibroblasts and / or fibroblast progenitor cells. Such co-culturing may be in serum-free or reduced-serum conditions.

[0316] In a further aspect, the invention provides a cellular composite comprising:

[0317] (a) a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0318] (b) a composition the composition comprising:

[0319] (i) myocytes and / or myocyte progenitor cells; and

[0320] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13: 1 to about 49: 1 ; wherein the cells of (i) and (ii) in the composition of (b) are located on and / or in the substrate of (a).

[0321] Suitably, the cellular substrate of (a) may further comprise adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1. The term “cellular composite” as used herein refers to an isolated artificial cell structure, i.e. not naturally occurring in the human or animal body.

[0322] The term “located on” the substrate as used herein refers to the cells of (i) and (ii) in the composition being directly placed above or below the substrate such that physical contact between the cellular substrate and cells of (i) and (ii) in the composition exists. In this context the substrate may be a 2D cellular substrate, for example a monolayer. The term “located in” the substrate as used herein refers to the cells of (i) and (ii) in the composition being placed within the substrate, for example within pores of the substrate. In this context the substrate may be a 3D cellular substrate.

[0323] In a further aspect, the present invention provides a method for generating a cellular composite that is suitable for use in cultivating meat in vitro, the method comprising:

[0324] (a) providing a composition, the composition comprising:

[0325] (i) myocytes and / or myocyte progenitor cells; and

[0326] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and

[0327] (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells to generate the cellular composite.

[0328] Suitably, the cellular substrate may further comprise adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

[0329] The various embodiments described herein relating to a method of cultivating animal biomass apply equally to the method for generating a cellular composite, unless required otherwise by the context.

[0330] In a suitable embodiment of the cellular composite or method of producing a cellular composite, the cellular substrate comprises fibroblasts. Suitably, the cellular substrate substantially consists of fibroblasts. Suitably, the fibroblasts may account for at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the cellular composite. In a suitable embodiment of the cellular composite or method of producing a cellular composite, the cellular substrate comprises fibroblasts and adipocytes. Suitably, the cellular substrate substantially consists of fibroblasts. Suitably, the fibroblasts may account for at least 60%, at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells in the cellular composite, with the adipocytes accounting for less than 10%.

[0331] Suitably, the cellular substrate comprises a cell monolayer or a tissue sheet.

[0332] Suitably, the cellular composite may be scaffold-free. By scaffold-free it is meant that the cellular composite does not comprise an artificially provided scaffold, such as a biocompatible scaffold. The cellular composite may, however, comprise a natively produced scaffold by the cells of the cellular composite. Such a scaffold may be for example extracellular matrix produced by the cells of the cellular composite.

[0333] The cellular composite of the present invention may comprise:

[0334] (i) myocytes and / or myocyte progenitor cells; and

[0335] (ii) adipocyte cells and / or adipose progenitor cells; and

[0336] (iii) fibroblast cells and / or fibroblast progenitor cells.

[0337] Optionally, the cellular composite may also comprise adipocytes or adipose progenitor cells.

[0338] In a further aspect, the present invention provides a composite produced by the method of generating a cellular composite as described herein.

[0339] The present invention also provided a method for cultivating meat in vitro in serum-free or reduced-serum conditions, the method comprising:

[0340] (a) providing a serum-free or reduced-serum composition, the composition comprising:

[0341] (i) myocytes and / or myocyte progenitor cells; and

[0342] (ii) adipocytes and / or adipose progenitor cells;

[0343] (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0344] (c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product.

[0345] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0346] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0347] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0348] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0349] EXAMPLES

[0350] Example 1 : Optimisation of muscle and fat cell co-culture ratios Materials and Methods

[0351] Media formulation

[0352] Cells and tissues were grown and maintained in medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) supplement, and 1% penicillin / streptomycin, alone (serum-free medium; SFM) or with macromolecular crowders: 10 g / L carrageenan (+Carr); 8.25 g / L polyethylene glycol MW 8,000 (+PEG8); 20 g / L polyethylene glycol MW 35,000 (+PEG35); 30 g / L polyvinylpyrrolidone MW 40,000 (+PVP40), and 10 g / L polyvinylpyrrolidone MW 360,000 (+PVP360). Serum-containing media comprising DMEM / F12 basal medium supplemented with 10% or 5% foetal bovine serum (FBS) and 1% penicillin / streptomycin were used as controls for cell proliferation and tissue production, respectively.

[0353] Cell proliferation assay in mono- and co-cultures

[0354] C2C12 mouse myoblasts (muscle cells; M) were seeded on polystyrene tissue culture plates alone (M100:F0; mono-culture) or combined with 3T3-F442A mouse pre-adipocytes (fat cells; F) mixed in a M99:F1 , M98:F2, M97:F3, M95:F5, M93:F7, and M90:F10 percent ratios (co-cultures) at a density of 5x103cells / cm2. Cells were grown for 5 days with serum-free media alone (SFM) or with macromolecular crowders (+Carr; +PEG8; +PEG35; +PVP40, or +PVP360), and with 10% FBS to evaluate the effects of co-culture on cell proliferation. Cells were incubated at 37°C and 5% CO2 in a humidified environment during cultures. Media was exchanged on day 1 , 3, and 5 of culture. All experiments were performed using three independent replicates.

[0355] Tissue production assay in mono- and co-cultures

[0356] C2C12 mouse myoblasts were seeded on polystyrene tissue culture plates alone (mono-cultures) or combined with 3T3-F442A mouse pre-adipocytes in a M95:F5 percent ratios (co-cultures) at a density of 1 *104cells / cm2, and then cultured for 14 days in serum-free (SFM), serum-containing (FBS), and serum-free, macromolecular crowder-supplemented (+PVP) conditions to evaluate the effects of co-culture on tissue production. Media was exchanged every 3-4 days of culture. All experiments were performed using three independent replicates.

[0357] Quantification of cell proliferation

[0358] Cell proliferation was evaluated at different time points via microscopy analysis of viable cells using the Calcein AM (live) / Propidium Iodide (dead) double cell staining, as well as via the resazurin metabolic analysis, with cell numbers calculated by interpolation using a fluorescence standard curve. All experiments were performed using three independent replicates, with proliferation expressed as percentage of seeded cells or as x-fold of SFM control).

[0359] Quantification of tissue formation

[0360] Total biomass production was evaluated via quantification of the wet weight of tissues formed by cells in different conditions after 14 days in culture, using a high-precision analytical balance. All experiments were performed using three independent replicates, with biomass expressed x-fold of SFM controls.

[0361] Statistical analysis

[0362] Error bars represent the standard deviation of the mean. Differences between groups were determined using one- or two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post hoc tests. Significance between groups was established for p < 0.05, 0.01 , and 0.001.

[0363] Results and Discussion The co-culture of fat cells (3T3-F442A mouse pre-adipocytes) within a larger population of muscle cells (C2C12 mouse myoblasts) showed surprising effects on the overall performance of the latter when grown with serum-free media (SFM) (Fig. 1).

[0364] In serum-free conditions, the co-culture of muscle and fat cells at ratios between M99:F1 and M97:F3 showed to have neutral effects on cell proliferation, whereas the M95:F5 seeding ratio resulted in the highest proliferation rates, with effects becoming apparent after 3 days in culture, and more obvious after 5 days in culture (Fig. 1a). In these conditions, co-cultures at a M93:F7 and M90:F10 did not improve cell proliferation.

[0365] The promoting effect of fat cells in co-culture was also augmented with the addition of macromolecular crowders to SFM (Fig. 1 b). In particular, cell proliferation in M95:F5 co-cultures was significantly improved over that of muscle cell mono-cultures in +PEG8, +PEG35, +PVP40, and +PVP360 conditions after 5 days in culture (Fig. 1 b). However, carrageenan did not impact cell proliferation beyond the effect of the co-culture itself.

[0366] The co-culture of C2C12 muscle cells with 3T3-F442A fat cells at M95:F5 ratio also resulted in improved tissue production yields both in serum-free and serum-containing conditions (Fig. 2). The proliferation of muscle cells grown for 14 days as mono-cultures benefited from the presence of serum (FBS) or of macromolecular crowder supplementation (+PVP), with corresponding significant increases in cell number (551±87% and 408±114% of SFM control, respectively) (Fig. 2a, white bars). Similarly, the deposition of extracellular matrix and tissue formation from muscle mono-cultures was significantly enhanced in FBS and +PVP conditions (264±83% and 421±64% of SFM control, respectively)(Fig. 2b, white bars). Muscle:fat co-cultures at a 95:5 ratio (grey bars) further augmented these effects, with cell proliferation in SFM, FBS, and +PVP increasing by 35%, 8%, and 17% over their corresponding mono-culture conditions (Fig. 2a). A similar impact was observed in terms of tissue production, with co-cultures in SFM, FBS, and +PVP showing a 64%, 50%, and 11% increase in biomass deposition over their corresponding mono-culture conditions (Fig. 2b). Importantly, the positive impact of muscle:fat co-culture was statistically significant in both SFM and FBS; moreover, co-cultures in +PVP were the best performing conditions tested, albeit increases from co-culture not being statistically significant (Fig. 2b).

[0367] Historically, the co-culture of fat cells with other cell types has been associated with poor culture performance, both in terms of cell proliferation and tissue formation. These inhibitions have been attributed to specific factors secreted by adipocytes that, when present in large amounts, become deleterious to the growth and metabolism of cells derived from muscle, skin, bone, and connective tissues, among others. However, such results have always involved the co-culture of fat cells in large proportions (i.e. , typically 50:50 percent ratios). In contrast, the present results showed that, in small proportions, fat cells can contribute to improve the performance of co-cultures, both in terms of cell proliferation and tissue biomass production, both in serum-free and serum-containing media. These improvements are likely still due to paracrine effects (that is, derived from the production and release of growth factors from adipocytes, but in concentrations that are beneficial instead of deleterious to muscle cells).

[0368] Example 2: Evaluation of tissue produced by muscle:fat co-cultures using high serum, low serum, and serum-free crowded media formulations

[0369] The inventors set out to evaluate porcine tissue growth using porcine muscle and / or fat cells in mono- and co-culture with a variety of media formulations and evaluate such conditions for porcine meat tissue formation and recovery.

[0370] Materials and Methods

[0371] Media formulation

[0372] Immortalised porcine skeletal muscle cells CSC-I2067Z (muscle cells) and corresponding tissues were grown as mono- or co-cultures with media comprising a base DMEM / F12 formulation with 4.5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate, and 1 % Insulin, Transferrin, Selenium , and Ethanolamine (ITS-X) supplement along with either 5% FBS (High- serum; control), 0.5% FBS and macromolecular crowders (Low-serum), or macromolecular crowders only (Serum-free). Primary porcine fat cell and tissue mono-cultures were also grown with High serum, Low serum, or Serum-free conditions, but the latter comprising a base DMEM / F12 formulation with 4.5mM GlutaMAX, 25 mM HEPES, 200 mg / L L-ascorbic acid 2- phosphate, 2% Insulin, Transferrin, Selenium , and Ethanolamine (ITS-X) 40 pg / L FGF2, 100 ng / L NRG1 , 100 ng / L TGFP3, 0.8 g / L BSA, and supplement macromolecular crowders.

[0373] Tissue production and recovery assays from muscle and fat mono- and co-cultures

[0374] Muscle and fat cells in High-serum medium were seeded on polystyrene tissue culture plates as mono- or co-cultures at a 95:5% ratio at a total density of 2*104cells / cm2and allowed to attach for 48h (mono-cultures) or 24h (co-cultures) at 37°C and 5% CO2 conditions. Cells were then washed with saline and cultured for 14 days with the various media formulations, at 37°C and 5% CO2 in a humidified environment, with media change every 3-4 days, in order to allow deposition of extracellular matrix and the formation of tissues. The density of the various tissues was evaluated via phase-contrast microscopy throughout the culture period up until collection. All experiments were performed using three independent replicates.

[0375] Quantification and qualification of produced tissues

[0376] Total biomass production was evaluated via quantification of the wet weight of tissues formed by cells in different conditions after 14 days in culture, using a high-precision analytical balance. Tissues were also analysed qualitatively via photography with background elimination in order to evaluate i) the ability of cells and tissues to remain attached to the surface throughout culture period, ii) the ability to recover tissues, iii) the integrity of recovered tissues, and iv) the ability to handle / process the tissues post-collection. All experiments were performed using three independent replicates, with biomass expressed x-fold of High-serum controls.

[0377] Statistical analysis

[0378] Error bars represent the standard deviation of the mean. Differences between groups were determined using one- or two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post hoc tests. Significance between groups was established for p < 0.05, 0.01 , and 0.001.

[0379] Results and discussion

[0380] The impact of muscle:fat co-culture on the quantity and quality of tissue production in various culture medium conditions was evaluated in terms of cell and tissue density (Fig. 3; Table 1) and tissue performance (Table 1).

[0381] Muscle mono-cultures maintained with High-serum were able to form tissues comprising dense concentrations of cells and extracellular matrix, however such tissues showed to lose clusters of cells during the later stages of culture, forming uneven tissue sheets due to localised cell contraction (Fig. 3, top left panel). These tissues remained firmly attached to the surfaces, were difficult to recover, and when recovered showed low integrity (friability) and handleability (Fig. 3, top left inset; Table 1). In Low-serum medium, muscle mono-cultures showed no cell contraction or detachment (Fig. 3, top centre panel). Tissues formed in these conditions remained attached to surfaces during culture, were easily recoverable, and showed good integrity and handleability (Fig. 3, top centre inset; Table 1). In contrast, Serum-free muscle mono-cultures showed similar performance to that of High-serum conditions, with the evident cell contraction and loss at later culture stages impacting tissue density, recovery, and integrity (Fig. 3, top right; Table 1).

[0382] Fat mono-cultures maintained with High-serum formed very thin tissue sheets comprising a dense layer of differentiating cells and extracellular matrix; however, cells showing larger intracellular accumulation of liquid droplets steadily lost adherence and detached from the underlying tissue (Fig. 3, middle left panel). These tissues remained firmly attached to the surfaces, were difficult to recover, and when recovered showed low integrity (friability) and handleability (Fig. 3, middle left inset; Table 1). In Low-serum medium, these effects were even more evident, with cultures showing sparser cell and tissue densities and continuous differentiated cell detachment (Fig. 3, middle centre panel). Tissues formed in these conditions were very difficult to recover and showed no structural integrity or handleability (Fig. 3, middle centre inset; Table 1). Fat mono-cultures maintained with Serum-free medium showed greater accumulation of cells compared with High- serum control conditions (Fig. 3, middle right panel), forming tissues that were easily recoverable, had minimal friability, but were still difficult to handle (Fig. 3, middle right inset; Table 1).

[0383] The addition of a small percentage of fat cells to muscle cells in co-culture resulted in the overall improvement of the production and performance of the resulting tissues. Co-cultures in High- serum formed dense tissues comprising several layers of cells and extracellular matrix, with minimal cell detachment throughout the culture period (Fig. 3, bottom left panel). In addition, the high degree of cell differentiation resulted in observable tissue contraction; however, and in contrast with tissues generated from muscle mono-cultures, this contraction did not compromise tissue integrity, but instead promoted tissue detachment after 14 days in culture, helping with tissue recovery (Fig. 3, bottom left inset; Table 1). Overall handleability of these tissues was still poor (Table 1). In Low-serum medium, muscle:fat co-cultures showed no cell contraction or detachment (Fig. 3, bottom centre panel), and formed very dense tissues that remained attached to surfaces during culture. Moreover, these tissues were easily recoverable, showing excellent robustness, integrity, and handleability (Fig. 3, bottom centre inset; Table 1). But most importantly, muscle:fat co-cultures were necessary and sufficient to allow the formation of dense, multistratified layers of tissue in serum-free conditions (Fig. 3, bottom right panel). Tissues generated this way showed minimal cell detachment or contraction during culture while demonstrating great integrity and robustness both pre- and post-collection, making them very easy to handle and process after recovery from growth surfaces (Fig. 3, bottom right inset; Table 1).

[0384] Table 1 : Performance of tissues produced from muscle and fat mono-cultures, and from muscle:fat cocultures in High-serum, Low-serum, and Serum-free media conditions. Tissue performance was evaluated qualitatively (yes / no) in terms of their ability to remain stably attached to the growth surface throughout the culture period, ease of recovery, structural integrity and ease of handling post-collection, and quantitatively in terms of biomass (mg of wet weight per cm2of growth surface).

[0385] Animal-free, serum-free conditions are fundamental requirements for the development and implementation of sustainable cellular agriculture processes, including for cultivated meat manufacture. As such, the poor performance of tissues generated from muscle and fat cell monocultures in serum-free media represent important limitations that need addressing. The co-culture of muscle and fat cells at the described ratios provides a simple and effective strategy to circumvent the multiple issues observed in serum-free conditions, including the easing of tissue recovery and processing, and the maximisation of tissue integrity and of biomass production yields.

[0386] Example 3: Evaluation of different co-culture strategies for muscle tissue formation

[0387] The inventors set out to evaluate the impact of various muscle:fat cell co-culture strategies on porcine muscle tissue formation and recovery, to understand if benefits from co-culture are dependent on spatial proximity between the different cell types.

[0388] Materials and Methods

[0389] Media formulation for porcine cell expansion and tissue formation

[0390] Immortalised porcine skeletal muscle cells CSC-I2067Z (muscle cells) were expanded with media comprising a base DMEM / F12 formulation with 4.5 mM L-alanyl-L-glutamine dipeptide, 5% FBS, 1 mM L-ascorbic acid 2-phosphate, and 1% Insulin, Transferrin, Selenium, and Ethanolamine (ITS-X) supplement (cell proliferation medium). Low-passage primary porcine fat cells were expanded with a similar medium formulation but containing 10% FBS instead. Tissues were generated from muscle and fat cell co-cultures with serum-free medium comprising a base DMEM / F12 formulation with 4.5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2- phosphate, 1% Insulin, Transferrin, Selenium, and Ethanolamine (ITS-X) supplement and 10 g / L polyvinylpyrrolidone MW 360,000 macromolecular crowder. Cells were incubated at 37°C and 5% CO2 in a humidified environment during cultures.

[0391] Co-culture strategies Muscle cells were seeded in co-culture with fat cells at a 95:5% ratio onto 25 cm2tissue culturegrade flasks at a total cell density of 1 x104cells / cm2using three distinct strategies (Fig. 4): i) homogeneous co-seeding, where muscle and fat cells were resuspended together with cell proliferation medium and seeded evenly and incubated for 1-48h at 37°C and 5% CO2 humidified conditions to form a homogeneous cell monolayer (Fig. 4a); ii) heterogeneous co-seeding, where 2.375x105muscle cells and 1.25X104fat cells were seeded in different regions of the dish and incubated for 1-48h at 37°C and 5% CO2 humidified conditions to form a monolayer of two spatially-separated cell populations (Fig. 4b); and iii) sequential seedings, where 2.375x105muscle cells were seeded and incubated for 1-48h at 37°C and 5% CO2 humidified conditions to form a homogeneous cell monolayer, and then subsequently seeded with 1.25X 104fat cells (Fig. 4c). After cell attachment was confirmed in all strategies, co-cultures were maintained with serum- free tissue production medium for up to 21 days, with medium change every 3-4 days, in order to allow deposition of extracellular matrix and production of biomass. Muscle mono-cultures were used as controls. The density, morphology, and behaviour of cells was evaluated via phasecontrast microscopy throughout the culture period. All experiments were performed using three independent replicates.

[0392] Quantification of cell proliferation

[0393] Cell proliferation was evaluated at day 2, 7, and 21 of culture via the resazurin metabolic analysis, with cell numbers calculated by interpolation using a fluorescence standard curve. All experiments were performed using three independent replicates, with proliferation expressed as percentage of seeded cells).

[0394] Quantification and qualification of produced tissues

[0395] Total biomass production was evaluated via quantification of the wet weight of tissues formed by cells in different conditions after 21 days in culture, using a high-precision analytical balance. All experiments were performed using three independent replicates, with biomass normalised against a standard (i.e. control tissue made from muscle mono-cultures).

[0396] Statistical analysis

[0397] Error bars represent the standard deviation of the mean. Differences between groups were determined using one- or two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post hoc tests. Significance between groups was established for p < 0.05, 0.01 , and 0.001.

[0398] Results and Discussion: The different cell types were easy to distinguish during the first 5 days in culture, independently of co-culture strategy, with the smaller muscle cells covering the growth substrate as disperse monolayers and the less numerous, larger fat cells tending to form dense clusters. These morphological differences were evident both in strategies where the two cell types were in close proximity, either side-by-side (i.e., homogeneous co-seeding) or top-to-bottom (i.e., sequential seedings), or where spatially separated (i.e., heterogeneous separation). After ? days, distinctions based on cell morphology were more difficult to ascertain due to the high density of confluent cell layers, and to cell displacement (e.g., from cell migration). No obvious interphase or separation was observed between the different cell populations, apart from the tendency of fat cells to grow and differentiate in clusters. After 21 days in culture, all tissues generated from co-culture strategies had a similarly dense, structurally robust appearance, comprising large accumulations of cells and extracellular matrix, and with no obvious heterogeneity to the naked eye. In contrast, tissues generated from control muscle mono-cultures were less dense and showed evident signs of cell detachment, contraction, and low structural integrity.

[0399] These observations were in line with results from cell quantification assays (Fig. 5). Specifically, cell number at day 2 was similar between different co-culture strategies and mono-culture controls and corresponded to 215-243% of original seeded cells (Fig. 5, white bars). At day 7, cell proliferation remained similar between co-cultures (i.e., 930-1062% of original seed), which was in turn higher than in control mono-cultures (784±165% of original seed), albeit not significantly so (Fig. 5, grey bars). However, at day 21 , all tissues generated from the three different co-culture strategies comprised significantly higher cell numbers (1326-1428% of original seed) compared to those from control muscle mono-cultures (701±225% of original seed) (Fig. 5; black bars).

[0400] These results illustrate that, in small ratios, the co-culture of fat cells improves the ability of muscle cells to remain viable and proliferate in serum-free conditions, and to subsequently form dense, structurally strong tissues. Furthermore, the similarity in performance between the different coculture strategies indicate that improvements do not appear to be dependent on the spatial proximity or direct interaction between the two cell types (e.g., via ligand-receptor or gap junction communication). This instead suggests that the benefits from co-cultures result from paracrine effects controlled by cell-specific soluble factors.

[0401] Example 4: Evaluation of co-cultures for enhanced skin cell proliferation and tissue formation The inventors set out to evaluate the ability of co-cultures of porcine skin and fat cells to accelerate overall cell proliferation and increase production of tissue biomass.

[0402] Materials and Methods

[0403] Cell proliferation assay in mono- and co-cultures

[0404] Primary dermal fibroblasts isolated from porcine skin and primary pre-adipocytes isolated from porcine fat were seeded at a density of 1 x104cells / cm2on polystyrene tissue culture plates in mono-cultures (100:0 and 0:100 ratio, respectively) or in co-cultures (99:1 , 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91 :9, 90:10, 85:15, and 80:20 percentage ratios) and incubated at 37°C and 5% CO2 in a humidified environment for 5 days with culture medium comprising DMEM / F12 with 5 mM L-alanyl-L-glutamine dipeptide, 0.25 mM L-ascorbic acid 2-phosphate salt, 1% Insulin- Transferrin-Selenium-Ethanolamine (ITS-X) supplement, 1.25 g / L recombinant human albumin, 25 pg / L IGF-1 , 2.5 pg / L FGF2, and 1.25 pg / L HGF. Medium was exchanged on day 1 , 3, and 5 of culture. Cell growth was evaluated at different time points via the resazurin metabolic analysis as well as by microscopy analysis, with cell numbers calculated by interpolation using a fluorescence standard curve. Experiments were performed seven independent times using three technical replicates each, with cell doubling time expressed as percentage normalised to the time of 100:0 control mono-cultures.

[0405] Tissue production assay in mono- and co-cultures

[0406] Primary dermal fibroblasts isolated from porcine skin and primary pre-adipocytes isolated from porcine fat were seeded at a density of 1 xio5cells / cm2on polystyrene tissue culture plates in mono-cultures (100:0 and 0:100 ratio, respectively) or in co-cultures (99:1 , 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91 :9, 90:10, 85:15, and 80:20 percentage ratios) and incubated at 37°C and 5% CO2 in a humidified environment up to 14 days with culture medium comprising DMEM / F12 with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate salt, 1% Insulin- Transferrin-Selenium-Ethanolamine (ITS-X) supplement, 1.25 g / L recombinant human albumin, 25 pg / L IGF-1 , 2.5 pg / L FGF2, 1.25 pg / L HGF and 4% London-mix™ macromolecular crowding supplement. Medium was exchanged every 3-4 days of culture. Biomass production was evaluated at day 7 and day 14 via quantification of total collagen content using the Picro-sirius Red assay as well as via quantification of total protein content using a modified Bradford assay. Experiments were performed seven independent times using three technical replicates each, with collagen and protein content expressed as percentage normalised to the content from 100:0 control tissues at day 7.

[0407] Statistical analysis Error bars represent the standard deviation of the mean. Differences between groups were determined using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post hoc tests. Significance between conditions was established for p < 0.05, 0.01 , and 0.001.

[0408] Results and Discussion

[0409] The co-culture of the pre-adipocytes within a larger population of fibroblasts showed surprising effects on the overall performance of the latter when grown with serum-free media (Fig. 6). In these conditions, the co-culture of skin and fat cells at ratios between 97:3 and 93:7 showed to promote overall cell proliferation by significantly reducing cell doubling time in a 5 day culture, whereas a larger proportion of fat cells (90:10 to 80:20 ratios) showed to impair overall proliferation by significantly increasing cell doubling time. Doubling time of fat cells in monoculture (0:100 ratio; Fig. 6, black bar) was almost twice that of skin cell alone (100:0 ratio; Fig. 6, white bar).

[0410] The promoting effect of fat cells in co-culture also improved skin tissue production yields, evaluated in terms of total collagen (Fig. 7A) and total protein content (Fig. 7B). Specifically, total collagen deposition at day 7 was shown to be significantly higher in skin:fat cell co-cultures with 98:2 to 91 :9 ratios compared with fibroblast mono-cultures (100:0 control), whereas the reverse was true in pre-adipocyte mono-cultures (0:100 control) (Fig. 7A, white bars). Collagen content was equally higher in tissues produced from co-cultures for 14 days, however only co-cultures with a 95:5 ratio was this increase significant when compared with the corresponding control (100:0 ratio at day 14) (Fig. 7A, grey bars). Similar trends were observed for total protein analysis, with tissues produced for 7 days from skin:fat cell co-cultures with 95:5 to 90:10 ratios showing a significantly higher protein content compared with fibroblast mono-cultures (100:0 control) (Fig. 7B, white bars), whereas in tissues produced for 14 days, this significant increase was observed from co-cultures with a 99:1 to 92:8 ratios (Fig. 7B, grey bars).

[0411] As previously mentioned, the co-culture of fat cells with fibroblasts has typically been investigated at 50:50 percent or similar ratios and has historically been associated with lower cell proliferation rates and worse tissue production yields. In contrast, the present results showed that, in small proportions, fat cells can contribute to improve skin cell proliferation and tissue biomass production in serum-free media, and these impacts are likely due to paracrine effects.

[0412] Example 5: Evaluation of muscle tissue formation using skin tissue substrates The inventors set out to evaluate porcine muscle tissue growth using porcine skin tissues as substrates for muscle and fat co-cultures and evaluate such substrates for porcine muscle tissue formation and recovery.

[0413] Materials and Methods

[0414] Media formulation for porcine cell expansion and tissue formation

[0415] Low-passage porcine primary skin (dermal) cells and porcine immortalised skeletal muscle cells CSC-I2067Z (muscle cells) were expanded with media comprising a base DMEM / F12 formulation with 4.5 mM L-alanyl-L-glutamine dipeptide, 5% FBS, 1 mM L-ascorbic acid 2-phosphate, and 1 % Insulin, Transferrin, Selenium, and Ethanolamine (ITS-X) supplement (cell proliferation medium). Low-passage porcine primary fat cells were expanded with a similar medium formulation but containing 10% FBS instead. Tissues were generated from skin or muscle cell mono-cultures, or muscle and fat, skin and muscle, or skin, muscle, and fat cell co-cultures with media comprising a base DMEM / F12 formulation with 4.5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate, 1% Insulin, Transferrin, Selenium, and Ethanolamine (ITS-X) supplement and macromolecular crowders (SFM) or trace-levels of serum (0.5% FBS) and macromolecular crowders (+FBS).

[0416] Production of skin tissue substrates

[0417] Low-passage porcine skin cells were seeded evenly on sterile tissue culture-grade dishes at a cell density of 1 *104cells / cm2and incubated with cell proliferation medium overnight to allow for attachment and formation of a cell monolayer. Skin cells were subsequently washed with sterile saline and cultured with SFM or +FBS tissue formation media for 7 days in order to allow the production of a confluent layer of skin tissue. Skin tissues were washed twice with saline prior to immediate use as substrates, or as skin-only control tissues. Cells were incubated at 37°C and 5% CO2 in a humidified environment during cultures.

[0418] Tissue production and recovery assays from mono- and co-cultures

[0419] Muscle cells were seeded alone or in co-culture with fat cells at a 95:5% ratio onto skin tissue substrates at a total cell density of 1 x104cells / cm2and incubated with cell proliferation medium at 37°C and 5% CO2 conditions for 4 h to allow for cell attachment. Cells on skin tissues were then washed with saline and cultured for 2 weeks with SFM or +FBS tissue formation media, with media change every 4 days, in order to allow deposition of extracellular matrix and production of biomass. Tissues made from skin or muscle cell mono-cultures, or muscle and fat co-cultures grown on tissue culture-grade dishes were similarly produced and used as controls. The density, morphology, and behaviour of cells was evaluated via phase-contrast microscopy throughout the culture period up until tissue collection. All experiments were performed using three independent replicates.

[0420] Quantification and qualification of produced tissues

[0421] Total biomass production was evaluated via quantification of the wet and dry weight of tissues formed by cells in different conditions after 2 weeks in culture, using a high-precision analytical balance. Tissues were also analysed qualitatively in order to evaluate i) the ability of cells and tissues to remain attached to the surface throughout culture period, ii) the ability to recover tissues, iii) the integrity of recovered tissues, and iv) the ability to handle / process the tissues postcollection. All experiments were performed using three independent replicates, with biomass expressed in absolute terms (mg) or normalised against a standard (i.e. control tissue made from muscle mono-cultures grown in SFM).

[0422] Statistical analysis

[0423] Error bars represent the standard deviation of the mean. Differences between groups were determined using one- or two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post hoc tests. Significance between groups was established for p < 0.05, 0.01 , and 0.001.

[0424] Results and Discussion:

[0425] Similarly to the experiments evaluating the impact of co-culturing small rations of fat cells on muscle cell proliferation and ability to form denser, more structurally robust tissues, this example explores the impact of using skin tissues as substrates for promoting the growth of muscle or muscle and fat cells and for achieving higher tissue biomass yields. Ultimately, the rationale behind the benefit of such substrates is three-fold: firstly, they provide a biological matrix that supports cell adhesion and prevents cell detachment during long-term cultures; secondly, they comprise a source of live dermal skin cells capable of acting as feeders, providing important trophic and signalling factors to nutrient-demanding cell types; and thirdly, they emulate the connective tissues from animal meat, and their presence in the final product represents additional edible, useful biomass that is safe for consumption.

[0426] As expected from other experiments, tissues generated from muscle mono-cultures in SFM showed signs of cell contraction, detachment and loss during later culture stages (Fig. 9(i)), which resulted in thinner, more fragile, difficult to recover tissues averaging wet and dry weight of 2.4±0.6 and 0.9±0.2 mg / cm2, respectively (Table 2, (i)). The presence of trace-levels of serum in culture improved tissue qualitative properties (Fig. 9(H)) and biomass over SFM conditions (Table 2, (ii)). Moreover, and as equally expected, muscle:fat co-cultures in SFM (Fig. 9(iii)) or +FBS (Fig. 9(iv)) further increased tissue quantity and quality over that of tissues produced from corresponding mono-cultures (Table 2, (iii) and (iv)).

[0427] Interestingly, muscle cells grown on skin tissue substrates showed to produce tissues both denser and more robust than those generated from muscle:fat co-cultures, with SFM conditions (Fig. 9(v)) resulting in better performance than with +FBS (Fig. 9(vi)), i.e. , 125% and 100% increase in dry weight over that of the control, respectively. Biomass yields were further increased in tissues produced from muscle:fat co-cultures grown on skin substrates, with SFM conditions again resulting in better performance than +FBS (Fig. 9(vii) and (viii); Table 2, (vii) and (viii), respectively).

[0428] Table 2. Both absolute and normalised wet and dry weight of tissues produced in various conditions, with values corresponding to the amount of tissue produced in a 1 cm2area. All values are expressed as the average and standard deviation of three independent repeats. Weight normalisation was performed against the average weight of tissues made from muscle cells grown on normal tissue culture surfaces in serum- free medium conditions (i.e., standard muscle tissue culture setup). Experiments (i) to (iv) used a non-tissue substrate (TC plastic), whereas (v) to (viii) used a skin tissue substrate; experiments (ix) and (x) represent the actual skin tissues substrates, cultured for the same period as in the other conditions.

[0429] The absence of trace amounts of serum typically limits muscle tissue formation, an effect particularly striking in tissues produced from muscle cells alone, and to a lesser degree, muscle and fat co-cultures. Inversely, muscle cells co-cultured with skin, or with fat and skin benefit from the absence of serum, with the highest tissue output being observed for the latter condition. Importantly, the advantage in having skin tissue in co-culture does not seem to be solely due to its contribution to overall biomass, as the dry weight of tissues produced from muscle:fat cells grown on skin (Table 2, (vii) and (viii) = 2.1 mg / cm2) is still higher than the dry weight of tissues from muscle:fat cells combined with that of skin substrates (Table 2, (iii) + (ix) = 1.5 mg / cm2; (iv) + (x) = 1.7 mg / cm2).

[0430] These results indicate that the best outcome, in terms of biomass production, was achieved when growing muscle and fat cells on skin substrates with macromolecular crowder-supplemented, serum-free medium. Overall, this system allowed to produce 4.3 mg of meat (wet weight) per cm2area, which corresponds to a 135% increase over the yield of tissues generated from muscle cells alone. In comparison, co-culturing muscle and fat resulted in a 35% increase, and muscle and skin in a 125% increase. Moreover, the confluent monolayer of dermal skin fibroblasts provided a rich extracellular matrix environment, along with cell signalling cues, for muscle and fat cells to differentiate and form myotubular tissue with integrated fat, allowing high quality, high biomass tissue to be produced.

[0431] The following numbered paragraphs (“para” or “paras”) also represent the invention as described herein.

[0432] Para 1. A method for cultivating meat in vitro, the method comprising:

[0433] (a) providing a composition, the composition comprising:

[0434] (i) myocytes and / or myocyte progenitor cells; and

[0435] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and

[0436] (b) co-culturing the cells in the composition to produce a cultivated meat product.

[0437] Para 2. The method of para 1 , wherein the co-culturing step is performed in serum-free or reduced-serum conditions.

[0438] Para 3. The method of any one of the preceding paras, wherein the co-culturing step is performed for at least 24 hours. Para 4. The method of any one of the preceding paras, wherein the cells of (i) and (ii) are seeded on a substrate at a cell density of at least 10,000 cells / cm2, or at least 5 cells / mm3.

[0439] Para 5. The method of any one of the preceding paras, wherein the cultivated meat product comprises a multicellular monolayer or a multicellular aggregate.

[0440] Para 6. A composition for cultivating meat in vitro, wherein the composition comprises:

[0441] (i) myocytes and / or myocyte progenitor cells; and

[0442] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, wherein the ratio is from about 13:1 to about 49:1.

[0443] Para 7. The composition of para 6, wherein the composition is a serum-free or reduced- serum composition.

[0444] Para 8. The method or composition of any one of the preceding paras, wherein the ratio is from about 19:1 to about 33:1.

[0445] Para 9. The method or composition of para 8, wherein the ratio is about 19:1.

[0446] Para 10. The method or composition of any one of the preceding paras, wherein the cells of (i) are muscle satellite cells and / or myoblasts.

[0447] Para 11. The method or composition of any one of the preceding paras, wherein the cells of (ii) are pre-adipocytes.

[0448] Para 12. The method or composition of any one of the preceding paras, wherein the composition comprises a cell culture medium.

[0449] Para 13. The method or composition of any one of paras 1 , 3 to 6, or 8 to 12, wherein the composition comprises serum.

[0450] Para 14. The method or composition of any one of paras 1 to 13, wherein the composition comprises a macromolecular crowding (MMC) agent. Para 15. The method or composition of para 14, wherein the MMC is selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

[0451] Para 16. The method or composition of para 14 or 15, wherein the MMC is selected from: PEG8, PEG35, PVP40, PVP360 and / or carrageenan; or combinations thereof.

[0452] Para 17. The method or composition of any one of paras 1 to 16, wherein the composition comprises L-glutamine, L-alanyl-L-glutamine dipeptide, a natural and / or synthetic peptide growth factor, a neuregulin, a morphogenic protein, a vitamin, a carrier molecule, an amino alcohol, a trace metal and / or a lipid, or an analogue thereof, or activator or inhibitor of their molecular pathways.

[0453] Para 18. The method or composition of any one of the preceding paras, wherein the composition is scaffold-free.

[0454] Para 19. A cultivated meat product produced by the method of any one of paras 1 to 5 or 8 to 18.

[0455] Para 20. A method for cultivating meat in vitro, the method comprising:

[0456] (a) providing a composition, the composition comprising:

[0457] (i) myocytes and / or myocyte progenitor cells; and

[0458] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ;

[0459] (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0460] (c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product.

[0461] Para 21. The method of para 20, wherein the co-culturing step is performed in serum-free or reduced-serum conditions.

[0462] Para 22. A cellular composite comprising:

[0463] (a) a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0464] (b) a composition the composition comprising: (i) myocytes and / or myocyte progenitor cells; and

[0465] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13: 1 to about 49: 1 ; wherein the cells of (i) and (ii) in the composition of (b) are located on and / or in the substrate of (a).

[0466] Para 23. A method for generating a cellular composite that is suitable for use in cultivating meat in vitro, the method comprising:

[0467] (a) providing a composition, the composition comprising:

[0468] (i) myocytes and / or myocyte progenitor cells; and

[0469] (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and

[0470] (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells to generate the cellular composite.

[0471] Para 24. The cellular composite of para 22, or the method of para 23, wherein the cellular composite is a serum-free or reduced-serum cellular composite.

[0472] Para 25. The method or cellular composite of any one of paras 20 to 24, wherein the cellular substrate comprises fibroblasts.

[0473] Para 26. The method or cellular composite of any one of paras 20 to 25, wherein the cellular substrate comprises a cell monolayer or a tissue sheet.

[0474] Para 27. The method or cellular composite of any one of paras 20 to 26, wherein the cells of (i) and (ii) in the composition of (b) are located on the substrate of (a).

[0475] Para 28. The method or cellular composite of any one of paras 20 to 27, wherein the composite is scaffold-free.

[0476] Para 29. A cellular composite produced by the method of any one of paras 23 to 29.

[0477] Para 30. A method for cultivating meat in vitro in or reduced-serum conditions, the method comprising: (a) providing a serum free serum-free or reduced-serum composition, the composition comprising:

[0478] (i) myocytes and / or myocyte progenitor cells; and

[0479] (ii) adipocytes and / or adipose progenitor cells; (b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and

[0480] (c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product. Para 31. The method of any one of paras 20, 21 , 23 to 28 or composition of any one of paras 22, or 24 to 28, wherein the cells of (i) are muscle satellite cells and / or myoblasts.

[0481] Para 32. The method of any one of paras 20, 21 , 23 to 28, 31 , or composition of any one of paras 22, or 24 to 28, or 31 , wherein the cells of (ii) are pre-adipocytes.

Claims

CLAIMS1. A method for cultivating animal biomass in vitro, the method comprising:(a) providing a composition comprising at least two cell populations, wherein the at least two cell populations are:(a1) (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49:1; or(a2) (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9:1 to about 99:1; and(b) co-culturing the cells in the composition to produce a cultivated animal biomass.

2. The method of claim 1 , wherein the method is for cultivating meat in vitro, and the method comprises:(a) providing a composition, the composition comprising:(i) myocytes and / or myocyte progenitor cells; and(ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and(b) co-culturing the cells in the composition to produce a cultivated meat product.

3. The method of claim 1, wherein the method comprises:(a) providing a composition, the composition comprising:(i) fibroblasts and / or fibroblast progenitor cells; and(ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9: 1 to about 99: 1 ; and(b) co-culturing the cells in the composition to produce a cultivated animal biomass.

4. The method of claim 3, wherein the cultivated animal biomass is cultivated meat, skin or hide.

5. The method of any one of the preceding claims, wherein the co-culturing step is performed in serum-free or reduced-serum conditions.

6. The method of any one of the preceding claims, wherein the co-culturing step is performed for at least 24 hours.

7. The method of any one of the preceding claims, wherein the cells of (i) and (ii) are seeded on a substrate at a cell density of at least 10,000 cells / cm2, or at least 5 cells / mm3.

8. The method of any one of the preceding claims, wherein the cultivated animal biomass comprises a multicellular monolayer or a multicellular aggregate.

9. A composition for cultivating animal biomass in vitro, wherein the composition comprises at least two cell populations, wherein the at least two cell populations are:(a1) (i) myocytes and / or myocyte progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, wherein the ratio is from about 13:1 to about 49:1 ; or(a2) (i) fibroblasts and / or fibroblast progenitor cells; and (ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9:1 to about 99:1.

10. The composition of claim 9, wherein the composition is for cultivating meat in vitro, wherein the composition comprises:(i) myocytes and / or myocyte progenitor cells; and(ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, wherein the ratio is from about 13:1 to about 49:1.11 . The method or composition of any one of the preceding claims, wherein the ratio is from about 19:1 to about 33:1.

12. The method or composition of claim 11 , wherein the ratio is about 19:1.

13. The method or composition of any one of the preceding claims, wherein myocytes and / or myocyte progenitor cells are muscle satellite cells and / or myoblasts.

14. The composition of claim 9, wherein the composition comprises:(i) fibroblasts and / or fibroblast progenitor cells; and(ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 9:1 to about 99:1.

15. The composition of claim 14, wherein animal biomass is cultivated meat, skin or hide.

16. The composition of claim 14 or 15, wherein the ratio is selected from the group consisting of:(i) about 97:3 to about 93:7;(ii) about 98:2 to about 91 :9;(iii) about 95:5 to about 9: 1 ; and(iv) about 99:1 to about 92:8.

17. The composition of claim 16, wherein the ratio is about 95:5.

18. The composition of any one of claims 9 to 17, wherein the composition is a serum-free or reduced-serum composition.

19. The method or composition of any one of the preceding claims, wherein the cells of (ii) are pre-adipocytes.

20. The method or composition of any one of the preceding claims, wherein the composition comprises a cell culture medium.

21. The method or composition of any one of claims 1 to 4, 6 to 17 and 19 to 20, wherein the composition comprises serum.

22. The method or composition of any one of claims 1 to 21 , wherein the composition comprises a macromolecular crowding (MMC) agent.

23. The method or composition of claim 22, wherein the MMC is selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

24. The method or composition of claim 22 or 23, wherein the MMC is selected from: PEG8, PEG35, PVP40, PVP360 and / or carrageenan; or combinations thereof.

25. The method or composition of any one of claims 1 to 24, wherein the composition comprises L-glutamine, L-alanyl-L-glutamine dipeptide, a natural and / or synthetic peptide growth factor, a neuregulin, a morphogenic protein, a vitamin, a carrier molecule, an amino alcohol, a trace metal and / or a lipid, or an analogue thereof, or activator or inhibitor of their molecular pathways.

26. The method or composition of any one of the preceding claims, wherein the composition is scaffold-free.

27. A cultivated animal biomass produced by the method of any one of claims 1 to 8 or 19 to 26.

28. A cultivated meat product produced by the method of any one of claims 1 to 2, 5 to 8 or 19 to 26.

29. A method for cultivating meat in vitro, the method comprising:(a) providing a composition, the composition comprising:(i) myocytes and / or myocyte progenitor cells; and(ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ;(b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and(c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product.

30. The method of claim 29, wherein the cellular substrate further comprises adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

31. The method of claim 30, wherein the co-culturing step is performed in serum-free or reduced-serum conditions.

32. A cellular composite comprising:(a) a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and(b) a composition the composition comprising:(i) myocytes and / or myocyte progenitor cells; and(ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13: 1 to about 49: 1 ; wherein the cells of (i) and (ii) in the composition of (b) are located on and / or in the substrate of (a).

33. The cellular composite of claim 32, wherein the cellular substrate of (a) further comprises adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

34. A method for generating a cellular composite that is suitable for use in cultivating meat in vitro, the method comprising:(a) providing a composition, the composition comprising:(i) myocytes and / or myocyte progenitor cells; and(ii) adipocytes and / or adipose progenitor cells; wherein (i) and (ii) are present in the composition at a ratio, and wherein the ratio is from about 13:1 to about 49: 1 ; and(b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells to generate the cellular composite.

35. The method of claim 34, wherein the cellular substrate further comprises adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

36. The cellular composite of claim 32 or 33, or the method of claim 34 or 35, wherein the cellular composite is a serum-free or reduced-serum cellular composite.

37. The method or cellular composite of any one of claims 29 to 36, wherein the cellular substrate comprises fibroblasts.

38. The method or cellular composite of any one of claims 29 to 37, wherein the cellular substrate comprises a cell monolayer or a tissue sheet.

39. The method or cellular composite of any one of claims 29 to 38, wherein the cells of the composition are located on the cellular substrate.

40. The method or cellular composite of any one of claims 29 to 39, wherein the composite is scaffold-free.41 . A cellular composite produced by the method of any one of claims 34 to 40.

42. A method for cultivating meat in vitro in or reduced-serum conditions, the method comprising:(a) providing a serum free serum-free or reduced-serum composition, the composition comprising:(i) myocytes and / or myocyte progenitor cells; and(ii) adipocytes and / or adipose progenitor cells;(b) contacting the composition with a cellular substrate comprising fibroblasts and / or fibroblast progenitor cells; and(c) co-culturing the cells in the composition with the cellular substrate to produce a cultivated meat product.

43. The method of claim 42, wherein the cellular substrate further comprises adipocytes and / or adipose progenitor cells, optionally wherein the (i) fibroblasts and / or fibroblast progenitor cells and (ii) adipocytes and / or adipose progenitor cells are present in the cellular substrate at a (i):(ii) ratio, and wherein the ratio is from about 9:1 to about 99:1.

44. The method or composition of any one of claims 29 to 43, wherein the myocytes and / or myocyte progenitor cells are muscle satellite cells and / or myoblasts.

45. The method or composition of any one of claims 29 to 44, wherein the adipocytes and / or adipose progenitor cells are pre-adipocytes.

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